Methods and Compositions for Poxvirus A35R Protein

ABSTRACT

The present invention provides methods and compositions for modulating an immune response in a subject, comprising administering to the subject an effective amount of an A35R protein or active fragment thereof of vaccinia virus or other poxvirus.

STATEMENT OF PRIORITY

This application is a divisional application of, and claims priority to,U.S. patent application Ser. No. 12/281,099, filed on Aug. 28, 2008 andissued as U.S. Pat. No. 8,202,521 on Jun. 19, 2012, which is a 35 U.S.C.§371 national phase application of PCT International Application No.PCT/US2006/007393, filed on Mar. 1, 2006, the entire contents of each ofwhich are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to poxvirus A35R protein, antibodiesthereto, and nucleic acids encoding the A35R protein and their use indiagnostic and therapeutic methods.

BACKGROUND ART

Poxviruses are large double-stranded DNA viruses with genomes that rangefrom 130 to 379 kbp. Poxviruses have worldwide distribution and infect awide variety of animals, including insects, birds and mammals. Since theeradication of smallpox and the cessation of vaccination programs,various poxvirus infections have begun to re-emerge clinicallyworldwide. Variola virus continues to present a bioterrorism threat andseveral other poxviruses infect humans, causing morbidity and mortality:molluscum contagiosum virus (MCV), monkeypox virus, Tanapox virus,Yaba-like disease virus, cowpox virus and Cantagalo virus (evolved froma vaccinia virus (VV) vaccine strain). The prevalence of poxviruses inanimals and humans and their propensity for recombination and geneacquisition suggest that it would be unwise to discount them asimportant human pathogens. This is especially true, since most emerginginfectious diseases are zoonoses, crossing from animals to humans, andpoxviruses are known to acquire mutations and become highly pathogenicin a new animal species.

The A35R gene is highly conserved in mammalian-tropic poxviruses. VV isthe model poxvirus to study the A35R protein (called A35R in Copenhagenstrain, 0158 in WR strain and a number of other designations in allmammalian-tropic poxviruses) and its role in the virus life cycle. Thepresent invention provides the discovery that the A35R protein hasimmunoregulatory activity. Thus, the present invention overcomesprevious shortcomings in the art by providing an A35R protein andbiologically active fragments thereof, as well as nucleic acids encodingthis protein and its fragments and antibodies and inhibitors specificthereto. These compositions are used, for example, in methods ofdiagnosing, treating and preventing infection by poxvirus, treating andpreventing other disorders and in modulating immune responses.

SUMMARY OF THE INVENTION

The present invention provides a method of modulating an immune responsein a subject, comprising administering to the subject animmunomodulating amount of an A35R protein or active fragment thereof ofvaccinia virus or other poxvirus and/or administering animmunomodulating amount of a nucleic acid encoding an A35R protein oractive fragment thereof of vaccinia virus or other poxvirus.

Further provided herein is a method of treating or preventing anautoimmune disorder in a subject, comprising administering to thesubject an effective amount of an A35R protein or active fragmentthereof of vaccinia virus or other poxvirus and/or administering aneffective amount of a nucleic acid encoding an A35R protein or activefragment thereof of vaccinia virus or other poxvirus.

In addition, the present invention provides a method of reducing thelikelihood of transplant rejection in a transplant recipient, comprisingadministering to the subject an effective amount of an A35R protein oractive fragment thereof of vaccinia virus or other poxvirus and/oradministering an effective amount of a nucleic acid encoding an A35Rprotein or active fragment thereof of vaccinia virus or other poxvirus.

Also included is a method of enhancing an immune response to a poxvirusvaccine in a subject, comprising administering to the subject animmunogenic amount of a poxvirus vaccine engineered to lack nucleic acidencoding an A35R protein and/or to lack A35R protein activity.

In additional embodiments, the present invention provides a method ofenhancing an immune response in a subject to a heterologous antigenencoded by a poxvirus vector, comprising administering to the subject animmunogenic amount of a poxvirus vector comprising nucleic acid encodingthe antigen and wherein the poxvirus vector is engineered to lacknucleic acid encoding an A35R protein and/or to lack A35R proteinactivity.

Furthermore, the present invention provides a method of treating orpreventing a detrimental immune response in a subject, comprisingadministering to the subject an effective amount of an A35R protein oractive fragment thereof of vaccinia virus or other poxvirus proteinand/or administering an effective amount of a nucleic acid encoding anA35R protein or active fragment thereof of vaccinia virus or otherpoxvirus.

A method is also provided herein of treating or preventing a detrimentalimmune response in a subject wherein the detrimental immune response iscaused by A35R protein activity, comprising administering to the subjectan effective amount of a substance that inhibits A35R protein activity.

The present invention further provides a method of enhancing animmunomodulating effect of a substance, comprising combining thesubstance with an A35R protein or active fragment thereof of vacciniavirus or other poxvirus and/or combining the substance with a nucleicacid encoding an A35R protein or active fragment thereof of vacciniavirus or other poxvirus.

Additionally provided herein is a method of treating or preventing apoxvirus infection in a subject, comprising administering to the subjectan effective amount of an inhibitor of A35R protein activity.

Further embodiments of this invention include a method of diagnosing apoxvirus infection in a subject, comprising: a) contacting a sample fromthe subject with an antibody that specifically binds A35R protein underconditions whereby an antigen/antibody complex can form; and b)detecting formation of the antigen/antibody complex, thereby diagnosingpoxvirus infection in a subject.

Also provided herein is a method of diagnosing poxvirus infection in asubject, comprising: a) contacting a sample from the subject with anA35R protein or antigenic fragment thereof under conditions whereby anantigen/antibody complex can form; and b) detecting formation of theantigen/antibody complex, thereby diagnosing poxvirus infection in asubject.

A method is also provided herein of detecting poxvirus in a sample,comprising: a) contacting the sample with an antibody that specificallybinds A35R protein under conditions whereby an antigen/antibody complexcan form; and b) detecting formation of the antigen/antibody complex,thereby detecting poxvirus in the sample.

Further provided herein is a method of detecting an antibody to poxvirusin a sample, comprising: a) contacting the sample with an A35R proteinor antigenic fragment thereof under conditions whereby anantigen/antibody complex can form; and b) detecting formation of theantigen/antibody complex, thereby detecting an antibody to poxvirus inthe sample.

In addition, the present invention provides a method of identifyingnucleic acid comprising a nucleotide sequence encoding A35R protein in asample, comprising: a) contacting the sample with an oligonucleotidecomprising a nucleotide sequence that is complementary to the nucleotidesequence encoding A35R protein, under conditions whereby nucleic acidhybridization can occur; and b) detecting nucleic acid hybridization,thereby detecting nucleic acid comprising the nucleotide sequenceencoding A35R protein in the sample.

Additional embodiments include a method of identifying nucleic acidcomprising a nucleotide sequence encoding A35R protein in a sample,comprising: a) contacting the sample with a pair of oligonucleotideprimers that are complementary to the nucleotide sequence encoding A35Rprotein, under conditions whereby nucleic acid amplification can occur;and b) detecting nucleic acid amplification, thereby detecting nucleicacid comprising the nucleotide sequence encoding A35R protein in thesample.

The present invention additionally provides a method of treating cancerin a subject, comprising administering to the subject a poxvirusengineered to lack A35R protein activity and in some embodiments, thepoxvirus can further comprise: a) nucleic acid encoding a costimulatorymolecule (e.g., B7.1, ICAM-1, LFA-3); b) nucleic acid encoding a tumorantigen; and/or c) nucleic acid encoding an immunomodulatory protein, inany combination.

In other embodiments, the present invention provides a compositioncomprising A35R protein and a pharmaceutically acceptable carrier and acomposition comprising a poxvirus engineered to lack A35R activity and apharmaceutically acceptable carrier and a composition consistingessentially of a nucleotide sequence encoding a poxvirus A35R proteinand a pharmaceutically acceptable carrier.

Various other objectives and advantages of the present invention willbecome apparent from the following detailed description.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the unexpected discovery that the A35Rprotein of poxvirus has immunomodulating activity. Thus, in oneembodiment, the present invention provides a method of modulating animmune response in a subject, comprising administering to the subject aneffective amount of an A35R protein or active fragment thereof ofvaccinia virus or other poxvirus and/or administering to the subject aneffective amount of a nucleic acid encoding an A35R protein or activefragment thereof of vaccinia virus or other poxvirus.

Further provided in this invention is a method of treating and/orpreventing an autoimmune disorder in a subject, comprising administeringto the subject an effective amount of an A35R protein or active fragmentthereof of vaccinia virus or other poxvirus and/or administering aneffective amount of a nucleic acid encoding an A35R protein or activefragment thereof of a vaccinia virus or other poxvirus. Nonlimitingexamples of autoimmune disorders that can be treated and/or prevented bythe methods of this invention include arthritis (e.g., rheumatoidarthritis or RA), multiple sclerosis (MS), diabetes (e.g., insulindependent diabetes mellitus or IDDM), systemic lupus erythematosus(SLE), myasthenia gravis, Crohns' disease, regional enteritis,vasculitis, ulcerative colitis, Sjogren's syndrome, ankylosingspondylitis, polymyositis and any other autoimmune disorder now known orlater identified. The methods of the present invention can further beemployed to treat allergies, allergic reactions, and any other diseaseor disorder associated with an aberrant and/or undesirable immuneresponse or reaction.

Additionally provided is a method of reducing the likelihood oftransplant rejection (or increasing the likelihood of successfultransplantation) in a transplant recipient, comprising administering tothe transplant recipient an effective amount of an A35R protein oractive fragment thereof of vaccinia virus or other poxvirus and/oradministering to the transplant recipient an effective amount of anucleic acid encoding an A35R protein or active fragment thereof ofvaccinia virus or other poxvirus. The reduction in the likelihood oftransplant rejection or increase in the likelihood of successfultransplantation is in comparison to the likelihood of transplantrejection or likelihood of successful transplantation in a transplantrecipient that did not receive an A35R protein or active fragmentthereof or a nucleic acid encoding an A35R protein or active fragmentthereof, as such likelihoods would be know and/or determined accordingto art-known standards. Furthermore, the protein, active fragment and/ornucleic acid of these methods can be administered to the transplantrecipient at any time relative to the transplantation (i.e., before,after and/or simultaneously, in any combination.

In additional embodiments of this invention, a method is provided ofenhancing an immune response to a poxvirus vaccine in a subject,comprising administering to the subject an immunogenic amount of apoxvirus-vaccine engineered to lack nucleic acid encoding an A35Rprotein and/or to lack A35R protein activity. Thus, the poxvirus used inthe vaccine can have a complete or partial deletion of the nucleotidesequence encoding the A35R protein or the poxvirus of the vaccine canhave an alteration in the nucleotide sequence encoding the A35R protein(e.g., a deletion, substitution, insertion, stop codon, missensemutation, nonsense mutation, or any other mutation), the end resultbeing a lack of A35R protein activity in a cell or subject to which thepoxvirus vaccine has been delivered or introduced. Nonlimiting examplesof poxviruses that can be altered according to the methods of thisinvention to lack A35R protein activity are the attenuated ModifiedVaccinia Ankara (MVA) vaccine, the DRYVAX vaccine, the Acambis vaccines(ACM1000 and ACM2000), as well as any other poxvirus vaccine now knownor later identified.

Also provided herein is a method of enhancing an immune response in asubject to a heterologous antigen encoded by a poxvirus vector,comprising administering to the subject an immunogenic amount of apoxvirus vector comprising nucleic acid encoding the antigen and whereinthe poxvirus vector is engineered to lack nucleic acid encoding an A35Rprotein and/or to lack A35R protein activity. As noted above, thepoxvirus used in the vaccine can have a complete or partial deletion ofthe nucleotide sequence encoding the A35R protein or the poxvirus of thevaccine can have an alteration in the nucleotide sequence encoding theA35R protein (e.g., a deletion, substitution, insertion, top codon,missense mutation, nonsense mutation, or any other mutation), the endresult being a lack of A35R protein activity in a cell or subject towhich the poxvirus vaccine has been delivered or introduced. The use ofpoxviruses as vectors to deliver antigens for vaccine is known in theart. Nonlimiting examples of such vectors include those described forrabies vaccines in Rupprecht et al. (Virus Research 111:101-105 (2005));HIV vaccines in Cebere et al. (Vaccine 24:417-425 (2006)); tuberculosisvaccines in Xing et al. (Curr. Gene Ther. 5:485-482 (2005)); and forcancer immunotherapies in Kaufman et al. (Curr. Gene Ther. 17:239-244(2006)).

In the methods provided herein for enhancing an immune response, such anenhancement is identified by comparison with an immune response in asubject that did not receive the protein, active fragment and/or nucleicacid of this invention. Such comparative studies can be carried outaccording to well known protocols in the art for detecting and/ormeasuring any number of immune responses. Nonlimiting examples of animmune response that can be enhanced by the methods of this inventioninclude antibody response (e.g., protective antibody response;neutralizing antibody response), cytotoxic T cell response, T helperresponse, interleukin-2 (IL-2) production; and vaccine efficacy.

Further provided herein is a method of treating or preventing adetrimental immune response in a subject, comprising administering tothe subject an effective amount of an A35R protein or active fragmentthereof of vaccinia virus or other poxvirus protein and/or an effectiveamount of a nucleic acid encoding an A35R protein or active fragmentthereof of a vaccinia virus or other poxvirus.

Also provided herein is a method of treating or preventing a detrimentalimmune response in a subject wherein the detrimental immune response iscaused by A35R protein activity, comprising administering to the subjectan effective amount of a substance that inhibits A35R protein activity.A substance that inhibits A35R activity, can be, but is not limited to aligand (e.g., an antibody or antibody fragment) that specifically bindsan A35R protein or active fragment thereof and/or a nucleic acid thatinhibits transcription or translation of nucleic acid encoding an A35Rprotein or active fragment thereof (e.g., an antisense nucleic acid thatbinds a coding sequence of the A35R protein, an interfering RNA thatinhibits or suppresses transcription and/or translation of the A35Rprotein, a ribozyme, etc.) Furthermore, small molecules and othercompounds and substances that inhibit the activity of A35R could be usedin the methods of this invention.

A detrimental immune response as described herein can be, for example, adetrimental immune response produced by an agent comprising the A35Rprotein or nucleic acid encoding the A35R protein. Examples of adetrimental immune response include but are not limited to allergicreaction or allergy, immune-mediated inflammation of the organs (e.g.,heart; central nervous system; kidney; liver), pathogen-inducedimmunopathology, autoimmune diseases and disorders (e.g., diabetes, SLE,MS), etc.

In other embodiments of this invention, the detrimental immune responsecan be an immune response produced by a bioterrorism agent (e.g., animmune response that is harmful to a subject that can be modulated bydelivery to the subject of the A35R protein or active fragment thereofand/or nucleic acid of this invention). Examples of such bioterrorismagents include but are not limited to infectious biological agents suchas pathogenic bacteria (e.g., Yersinia causing plague; Bacillusanthracia causing anthrax; Francisella tularensis), viruses (e.g.,influenza; SARS coronavirus; Ebola virus; Marburg virus), toxins, etc.,as well as any other agent now known or later identified that can beused as a bioterrorism agent.

In yet other embodiments, the detrimental immune response can beproduced by the presence, either naturally or unnaturally, in thebioterrorism agent, of the A35R protein or active fragment thereof or anucleic acid encoding the A35R protein or active fragment thereof. Insuch embodiments, the detrimental immune response is treated by deliveryto the subject of a substance as described herein that inhibits orreduces A35R protein activity. Examples of such bioterrorism agents caninclude smallpox, vectors comprising A35R activity and other infectiousagents and toxic agents as described herein that are engineered to haveA35R activity.

In yet further embodiments of this invention, a method is provided ofenhancing an immunomodulating effect of a substance, comprisingcombining the substance with an A35R protein or active fragment thereofof vaccinia virus or other poxvirus and/or combining the substance witha nucleic acid encoding an A35R protein or active fragment thereof. Sucha substance that has an immunomodulating effect can be but is notlimited to steroids, immunosuppressive drugs, interferons,corticosteroids, azathioprine, cyclophosphamide, prednisone,methotrexate, rituximab, etc., as well as any other immunomodulatingagent now known or later identified.

An enhancement of an immunomodulating effect of a substance would beidentified by comparison of an immunomodulating effect of the substancein a subject with and without the presence of the A35R protein or activefragment thereof or nucleic acid encoding the A35R protein or activefragment thereof. As used herein, an “immunomodulating effect” is anyaction or activity of a cell or tissue of the immune system. Such animmunomodulating effect can be positive or negative, an enhancement oran inhibition, an increase or a decrease in a response, activity and/orreaction. Ways to detect and/or measure an immunomodulating effect areknown in the art and include standard protocols such as those used todetect and/or measure antibody production or activity, T lymphocyteproliferation or activity, cytotoxicity and/or cytokine production oractivity.

The present invention additionally provides a method of treating orpreventing a poxvirus infection in a subject, comprising administeringto the subject an effective amount of an inhibitor of A35R proteinactivity. As noted above, a substance that inhibits A35R activity, canbe, but is not limited to a ligand, an antibody or an antibody fragmentthat specifically binds an A35R protein or active fragment thereof, anucleic acid that inhibits transcription or translation of nucleic acidencoding an A35R protein or active fragment thereof and/or a drug orother substance that acts to inhibit A35R activity (e.g.,immunosuppressive drugs, steroids, interferon, etc.)

The method of treating or preventing infection caused by poxvirus in asubject can be carried out, for example, by contacting an immune cell ofthe subject with any of the polypeptides, fragments, nucleic acids,vectors and/or antibodies of this invention. The cell can be, forexample, a CD8⁺ T cell which is contacted with the polypeptide and/orfragment of this invention in the presence of a class I MHC molecule,which can be a soluble molecule or it can be present on the surface of acell which expresses class I MHC molecules. The cell can also be anycell that can take up and express exogenous nucleic acid and produce thepolypeptides and/or fragments of this invention. In some embodiments,the polypeptides and/or fragments of this invention can be produced by acell that secretes them, whereby the polypeptide and/or fragment isproduced and secreted and then taken up and subsequently processed by anantigen presenting cell or other class I MHC-expressing cell andpresented to the immune system for induction of an immune response. Inother embodiments, the nucleic acids and/or vectors of this inventioncan be directly introduced into an antigen presenting cell and/or otherclass I MHC-expressing cell in which the polypeptide and/or fragment isproduced and processed directly and presented to the immune system onthe cell surface.

As set forth above, it is contemplated that in the methods wherein thecompositions of this invention are administered to a subject or to acell of a subject, such methods can further comprise the step ofadministering a suitable adjuvant to the subject or to a cell of thesubject. The adjuvant can be in the composition of this invention or theadjuvant can be in a separate composition comprising the suitableadjuvant and a pharmaceutically acceptable carrier. The adjuvant can beadministered prior to, simultaneous with, and/or after administration ofthe composition containing any of the polypeptides, fragments, nucleicacids and/or vectors of this invention. For example, QS-21, similar toalum, complete Freund's adjuvant, SAF, etc., can be administered withindays/weeks/hours (before or after) of administration of the compositionof this invention. The effectiveness of an adjuvant can be determined bymeasuring the immune response directed against the polypeptide and/orfragment of this invention with and without the adjuvant, using standardprocedures, as described herein and as are well known in the art.

The subject of this invention can be any subject in need of the immuneresponse of this invention and/or in need of treatment for or preventionfrom poxvirus infection, as well as any subject in whom it is desirableto induce an immune response to poxvirus. Such a subject can be any typeof animal that is susceptible to infection by a poxvirus of thisinvention, as well as any animal to which the proteins, active fragmentsthereof and nucleic acids of this invention can be administeredaccording to the methods of this invention. For example, an animal ofthis invention can be a mammal, a bird or a reptile. In certainembodiments, the subject of this invention is a human.

Symptoms of poxvirus infection can include fever, headache, muscleand/or joint aches, fever, rash, inflammation, etc. Appropriatetreatment can lead to a reduction in the severity of and/or eliminationof one or more of these symptoms.

The compositions of this invention can be administered to a cell of asubject or to a subject either in vivo or ex vivo. For administration toa cell of the subject in vivo, as well as for administration to thesubject, the compositions of this invention can be administered orally,parenterally (e.g., intravenously), by intramuscular injection, byintraperitoneal injection, subcutaneous injection, transdermally,extracorporeally, topically or the like. Also, the compositions of thisinvention can be pulsed onto dendritic cells, which are isolated orgrown from a subject's cells, according to methods well known in theart, or onto bulk peripheral blood mononuclear cells (PBMC) or variouscell subfractions thereof from a subject.

The exact amount of the composition required will vary from subject tosubject, depending on the species, age, weight and general condition ofthe subject, the particular composition used, its mode of administrationand the like. Thus, it is not possible to specify an exact amount forevery composition of this invention. However, effective amount can bedetermined by one of ordinary skill in the art using only routineexperimentation given the teachings herein.

As an example, to a subject diagnosed with poxvirus infection or knownto be at risk of being infected with poxvirus or in whom it is desirableto induce an immune response to poxvirus, between about 0.1 μg/kg toabout 10 g/kg of a polypeptide and/or biologically active fragment ofthis invention can be administered subcutaneously and can be in anadjuvant, at hourly, daily and/or weekly intervals until an evaluationof the subject's clinical parameters indicate that the subject isrecovered from infection by poxvirus and/or the subject demonstrates thedesired immunological response. Alternatively, a polypeptide and/orfragment of this invention can be pulsed onto dendritic cells at aconcentration of between about 0.1 ng to about 500 mg and the dendriticcells can be administered to the subject intravenously at the same timeintervals. The treatment can be continued or resumed if the subject'sclinical parameters indicate that poxvirus infection is present and canbe maintained until the infection is no longer detected by theseparameters and/or until the desired immunological response is achieved.

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the subject's body according to standard protocolswell known in the art. The polypeptides and/or biologically activefragments of this invention can be introduced into the cells via knownmechanisms for uptake of polypeptides into cells (e.g., phagocytosis,pulsing onto class I MHC-expressing cells, liposomes, etc.). The cellscan then be infused (e.g., in a pharmaceutically acceptable carrier) ortransplanted back into the subject per standard methods for the cell ortissue type. Standard methods are known for transplantation or infusionof various cells into a subject.

The pharmaceutical compositions of this invention include those suitablefor oral, rectal, topical, inhalation (e.g., via an aerosol) buccal(e.g., sub-lingual), vaginal, parenteral (e.g., subcutaneous,intramuscular, intradermal, intraarticular, intrapleural,intraperitoneal, intracerebral, intraarterial, or intravenous), topical(i.e., both skin and mucosal surfaces, including airway surfaces) andtransdermal administration, although the most suitable route in anygiven case will depend, as is well known in the art, on such factors asthe species, age, gender and overall condition of the subject, thenature and severity of the condition being treated and/or on the natureof the particular composition (i.e., dosage, formulation) that is beingadministered.

Pharmaceutical compositions suitable for oral administration can bepresented in discrete units, such as capsules, cachets, lozenges, ortables, each containing a predetermined amount of the composition ofthis invention; as a powder or granules; as a solution or a suspensionin an aqueous or non-aqueous liquid; or as an oil-in-water orwater-in-oil emulsion. Oral delivery can be performed by complexing acomposition of the present invention to a carrier capable ofwithstanding degradation by digestive enzymes in the gut of an animal.Examples of such carriers include plastic capsules or tablets, as knownin the art. Such formulations are prepared by any suitable method ofpharmacy, which includes the step of bringing into association thecomposition and a suitable carrier (which may contain one or moreaccessory ingredients as noted above). In general, the pharmaceuticalcomposition according to embodiments of the present invention areprepared by uniformly and intimately admixing the composition with aliquid or finely divided solid carrier, or both, and then, if necessary,shaping the resulting mixture. For example, a tablet can be prepared bycompressing or molding a powder or granules containing the composition,optionally with one or more accessory ingredients. Compressed tabletsare prepared by compressing, in a suitable machine, the composition in afree-flowing form, such as a powder or granules optionally mixed with abinder, lubricant, inert diluent, and/or surface active/dispersingagent(s). Molded tablets are made by molding, in a suitable machine, thepowdered compound moistened with an inert liquid binder.

Pharmaceutical compositions suitable for buccal (sub-lingual)administration include lozenges comprising the composition of thisinvention in a flavored base, usually sucrose and acacia or tragacanth;and pastilles comprising the composition in an inert base such asgelatin and glycerin or sucrose and acacia.

Pharmaceutical compositions of this invention suitable for parenteraladministration can comprise sterile aqueous and non-aqueous injectionsolutions of the composition of this invention, which preparations arepreferably isotonic with the blood of the intended recipient. Thesepreparations can contain anti-oxidants, buffers, bacteriostats andsolutes, which render the composition isotonic with the blood of theintended recipient. Aqueous and non-aqueous sterile suspensions,solutions and emulsions can include suspending agents and thickeningagents. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like.

The compositions can be presented in unit\dose or multi-dose containers,for example, in sealed ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example, saline or water-for-injectionimmediately prior to use.

Extemporaneous injection solutions and suspensions can be prepared fromsterile powders, granules and tablets of the kind previously described.For example, an injectable, stable, sterile composition of thisinvention in a unit dosage form in a sealed container can be provided.The composition can be provided in the form of a lyophilizate, which canbe reconstituted with a suitable pharmaceutically acceptable carrier toform a liquid composition suitable for injection into a subject. Theunit dosage form can be from about 1 μg to about 10 grams of thecomposition of this invention. When the composition is substantiallywater-insoluble, a sufficient amount of emulsifying agent, which isphysiologically acceptable, can be included in sufficient quantity toemulsify the composition in an aqueous carrier. One such usefulemulsifying agent is phosphatidyl choline.

Pharmaceutical compositions suitable for rectal administration arepreferably presented as unit dose suppositories. These can be preparedby admixing the composition with one or more conventional solidcarriers, such as for example, cocoa butter and then shaping theresulting mixture.

Pharmaceutical compositions of this invention suitable for topicalapplication to the skin preferably take the form of an ointment, cream,lotion, paste, gel, spray, aerosol, or oil. Carriers that can be usedinclude, but are not limited to, petroleum jelly, lanoline, polyethyleneglycols, alcohols, transdermal enhancers, and combinations of two ormore thereof. In some embodiments, for example, topical delivery can beperformed by mixing a pharmaceutical composition of the presentinvention with a lipophilic reagent (e.g., DMSO) that is capable ofpassing into the skin.

Pharmaceutical compositions suitable for transdermal administration canbe in the form of discrete patches adapted to remain in intimate contactwith the epidermis of the subject for a prolonged period of time.Compositions suitable for transdermal administration can also bedelivered by iontophoresis (see, for example, Pharmaceutical Research3:318 (1986)) and typically take the form of an optionally bufferedaqueous solution of the composition of this invention. Suitableformulations can comprise citrate or bis\tris buffer (pH 6) orethanol/water and can contain from 0.1 to 0.2M active ingredient.

The present invention further provides a medicament and the preparationthereof for use in treating and/or preventing the disease and disordersdescribed herein by employing the same steps as described in the methodsdisclosed herein. Also provided herein is a medicament and thepreparation thereof for use in modulating (enhancing, treating orpreventing a detrimental immune response, inhibiting, etc.) an immuneresponse according to the steps of the methods described herein. It isfurther contemplated that the methods, compositions and medicaments ofthis invention can be used for veterinary application as well as inapplications involving humans.

Furthermore, the nucleic acids and vectors of this invention can beadministered orally, intranasally, parenterally (e.g., intravenously),by intramuscular injection, by intraperitoneal injection, transdermally,extracorporeally, topically or the like. In the methods described hereinwhich include the administration and uptake of exogenous DNA into thecells of a subject (i.e., gene transduction or transfection), thenucleic acids of the present invention can be in the form of naked DNAor the nucleic acids can be in a vector for delivering the nucleic acidsto the cells for expression of the polypeptides and/or fragments of thisinvention. The vector can be a commercially available preparation or canbe constructed in the laboratory according to methods well known in theart.

Delivery of the nucleic acid or vector to cells can be via a variety ofmechanisms. As one example, delivery can be via a liposome, usingcommercially available liposome preparations such as LIPOFECTIN,LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.), SUPERFECT (Qiagen,Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison,Wis.), as well as other liposomes developed according to proceduresstandard in the art. In addition, the nucleic acid or vector of thisinvention can be delivered in vivo by electroporation, the technologyfor which is available from Genetronics, Inc. (San Diego, Calif.) aswell as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp.,Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system, which can package a recombinant retroviralgenome. The recombinant retrovirus can then be used to infect andthereby deliver to the infected cells nucleic acid encoding thepolypeptide and/or fragment of this invention. The exact method ofintroducing the exogenous nucleic acid into mammalian cells is, ofcourse, not limited to the use of retroviral vectors. Other techniquesare widely available for this procedure including the use of adenoviralvectors, alphaviral vectors, adeno-associated viral (AAV) vectors,lentiviral vectors, pseudotyped retroviral vectors and vaccinia viralvectors, as well as any other viral vectors now known or developed inthe future. Physical transduction techniques can also be used, such asliposome delivery and receptor-mediated and other endocytosismechanisms. This invention can be used in conjunction with any of theseor other commonly used gene transfer methods.

As one example, the nucleic acid of this invention is delivered to thecells of a subject in a vaccinia virus vector, the dosage foradministration of vaccinia virus to humans can typically range fromabout 10⁴ to 10⁹ pfu per injection, but in some embodiments, can be ashigh as 10¹², 10¹⁵ and/or 10²⁰ pfu per injection.

As another example, if the nucleic acid of this invention is deliveredto the cells of a subject in an adenovirus vector, the dosage foradministration of adenovirus to humans can range from about 10⁴ to 10⁹plaque forming units (pfu) per injection, but can be as high as 10²⁰10¹², 10¹⁵ and/or 10²⁰ pfu per injection.

In some embodiments, a subject will receive a single injection of aviral vector comprising a nucleic acid of this invention. If additionalinjections are necessary, they can be repeated at daily/weekly/monthlyintervals for an indefinite period and/or until the efficacy of thetreatment has been established. As set forth herein, the efficacy oftreatment can be determined by evaluating the symptoms and clinicalparameters described herein and/or by detecting a desired immunologicalresponse.

The exact amount of the nucleic acid or vector required will vary fromsubject to subject, depending on the species, age, weight and generalcondition of the subject, the particular nucleic acid or vector used,its mode of administration and the like. Thus, it is not possible tospecify an exact amount for every nucleic acid or vector. However, anappropriate amount can be determined by one of ordinary skill in the artusing only routine experimentation given the teachings herein.

The present invention further provides a method of diagnosing a poxvirusinfection in a subject, comprising: a) contacting a sample from thesubject with an antibody that specifically binds an A35R protein underconditions whereby an antigen/antibody complex can form; and b)detecting formation of the antigen/antibody complex, thereby diagnosingpoxvirus infection in a subject.

Also provided herein is a method of diagnosing poxvirus infection in asubject, comprising: a) contacting a sample from the subject with anA35R protein or antigenic fragment thereof under conditions whereby anantigen/antibody complex can form; and b) detecting formation of theantigen/antibody complex, thereby diagnosing poxvirus infection in asubject.

In other embodiments, a method is provided of detecting poxvirus in asample, comprising: a) contacting the sample with an antibody thatspecifically binds A35R protein under conditions whereby anantigen/antibody complex can form; and b) detecting formation of theantigen/antibody complex, thereby detecting poxvirus in the sample.

Further provided is a method of detecting an antibody to poxvirus in asample, comprising: a) contacting the sample with an A35R protein orantigenic fragment thereof under conditions whereby an antigen/antibodycomplex can form; and b) detecting formation of the antigen/antibodycomplex, thereby detecting an antibody to poxvirus in the sample.

The present invention provides a method of detecting a nucleic acidcomprising a nucleotide sequence encoding A35R protein in a sample,comprising: a) contacting the sample with an oligonucleotide comprisinga nucleotide sequence that is complementary to the nucleotide sequenceencoding A35R protein, under conditions whereby nucleic acidhybridization can occur; and b) detecting nucleic acid hybridization,thereby detecting nucleic acid comprising the nucleotide sequenceencoding A35R protein in the sample.

Additionally provided is a method of detecting nucleic acid comprising anucleotide sequence encoding A35R protein in a sample, comprising: a)contacting the sample with a pair of oligonucleotide primers that arecomplementary to the nucleotide sequence encoding A35R protein, underconditions whereby nucleic acid amplification can occur; and b)detecting nucleic acid amplification, thereby detecting nucleic acidcomprising the nucleotide sequence encoding A35R protein in the sample.

A sample of this invention can be any sample in which poxvirus proteinsand/or nucleic acids can be present. For example, the sample can be abody fluid, cells or tissue, including but not limited to, blood, serum,plasma, saliva, sputum, broncheoalveolar lavage, urine, semen, jointfluid, cerebrospinal fluid and cells, fluids and/or tissue from allorgans to which poxvirus antigens can disseminate including lung, liver,heart, brain, kidney, spleen, muscle, etc. A sample of this inventioncan also include a substance not obtained from the body of a subject ofthis invention. Examples of such a sample include but are not limitedto, a water sample, a food or foodstuff sample, a plant or plantmaterial sample, a soil or dirt sample, an effluent sample, etc.

In further embodiments, the compositions of this invention can also beused to treat or prevent cancer in a subject. Thus, in additionalembodiments, the present invention provides a method of treating cancerin a subject, comprising administering to the subject a poxvirusengineered to lack A35R protein activity. A poxvirus employed in thesemethods would be a poxvirus that is specific for cancer cells.

In other embodiments, the present invention provides a method oftreating cancer in a subject, comprising administering to the subject apoxvirus engineered to lack A35R protein activity and wherein thepoxvirus can further comprise a) nucleic acid encoding a costimulatorymolecule (e.g., B7.1, ICAM-1, LFA-3, etc., as known in the art); b)nucleic acid encoding a tumor antigen; and c) nucleic acid encoding animmunomodulatory protein or molecule. These nucleic acids can be presentseparately and/or in any combination in the poxvirus genome.

The present invention further provides compositions. Thus, in oneembodiment, provided herein is a composition comprising A35R protein anda pharmaceutically acceptable carrier. In other embodiments, a poxvirusengineered to lack A35R activity is provided. Further provided is acomposition comprising a poxvirus engineered to lack A35R activity and apharmaceutically acceptable carrier. Also provided herein is an isolatednucleic acid encoding an A35R protein or active fragment thereof of avaccinia virus or other poxvirus. Additionally provided herein is acomposition comprising an isolated nucleic acid encoding an A35R proteinor active fragment thereof of a vaccinia virus and a pharmaceuticallyacceptable carrier. In further embodiments, the present inventionprovides a composition comprising an antibody that specifically binds anA35R protein or active fragment thereof and a pharmaceuticallyacceptable carrier, as well as an antibody that specifically binds anA35R protein or active fragment thereof.

As indicated herein, the present invention provides biologically activefragments of the proteins of this invention, as well as antibodies thatspecifically bind the proteins and/or fragments of the proteins of thisinvention.

Further provided are isolated nucleic acids comprising, consistingessentially of and/or consisting of nucleotide sequences that encode theproteins and fragments of this invention. In particular, the presentinvention provides an isolated nucleic acid comprising, consistingessentially of, and/or consisting of the nucleotide sequence of Vacciniavirus Copenhagen strain (SEQ ID NO:1):

atggacgccgcgtttgttattactccaatgggtgtgttgactataacagatacattgtatgatgatctcgatatttcaatcatggactttataggaccatacattataggtaacataaaaactgtccaaatagatgtacgggatataaaatattccgacatgcaaaaatgctactttagctataagggtaaaatagttcctcaggattctaatgatttggctagattcaacatttatagcatttgtgccgcatacagatcaaaaaataccatcatcatagcatgcgactatgatatcatgttagatatagaagataaacatcagccattttatctattcccatctattgatgtttttaacgctacaatcatagaagcgtataacctgtatacagctggagattatcatctaatcatcaatccttcagataatctgaaaatgaaattgtcgtttaattcttcattctgcatatcagacggcaatggatggattataattgatgggaaatgcaatagtaaattttttatca.

Further provided herein is the nucleotide sequence of the A35R proteinof all of the poxviruses identified in Table 1 by virus name, gene namefor the A35R ortholog, the GenBank® database accession number, thenumber of amino acids and the location of the start and stop sites ofthe open reading frame (ORF) for A35R. Each of these nucleotidesequences is incorporated by reference herein in their entirety.

Additionally provided is a nucleic acid comprising, consistingessentially of, and/or consisting of a nucleotide sequence that encodesan amino acid sequence comprising, consisting essentially of, and/orconsisting of the amino acid sequence or a biologically active fragmentof any of the following amino acid sequences:

Vaccinia virus Copenhagen strain A35R (SEQ ID NO: 2):MDAAFVITPMGVLTITDTLYDDLDISIMDFIGPYIIGNIKTVQIDVRDIKYSDMQKCYFSYKGKIVPQDSNDLARFNIYSICAAYRSKNTIIIACDYDIMLDIEDKHQPFYLFPSIDVFNATIIEAYNLYTAGDYHLIINPSDNLKMKLSFNSSFCISDGNGWIIIDGKCNSNFLS;Variola virus strain Garcia A39R gene/protein: (SEQ ID NO: 3):MDTTFVITPMGMLTITDTLYDDLDISIMDFIGPYIIGNIKTVQIDVRDIKYSDMQKCYFS; andVariola virus strain India A38R: (SEQ ID NO: 4):MDTTFVITPMGMLTITDTLYDDLDISIMDFIGPYIIGNIKTVQIDVRDIKYSDMQKCYFS

Further provided herein is a nucleic acid that is the complement of eachand any of the nucleic acids of this invention.

The present invention also provides an isolated A35R protein, which canbe a protein having the amino acid sequence of SEQ ID NO:2. Alsoprovided herein is the amino acid sequence for the A35R protein of thepoxviruses identified in Table 1, as available in the GenBank® database.These amino acid sequences are incorporated by reference herein in theirentirety. Specific examples of nucleotide sequences of poxviruses ofthis invention, such as Bovine popular stomatitis virus (SEQ ID NO:8),Camelpox virus (SEQ ID NO:9), Cowpox virus (SEQ ID NO:10), Deerpox virus(SEQ ID NO:11), Ectromelia virus (SEQ ID NO:12), Goatpox virus Pellor(SEQ ID NO:13), lumpy skin disease virus (SEQ ID NO:14), Molluscumcontagiosum virus (SEQ ID NO:15), Monkeypox virus (SEQ ID NOS:16, 17 and18), Myxoma virus (SEQ ID NO:19), Orf virus (SEQ ID NO:20), Rabbitpoxvirus (SEQ ID NO:21), Rabbit fibroma virus (SEQ ID NO:22), sheeppoxvirus (SEQ ID NO:23), Swinepox virus (SEQ ID NO:24), Vaccinia virus (SEQID NOS:25, 26, 27, 28 and 29), Yaba-like disease virus (SEQ ID NO:30)and Yaba-like monkey tumor virus (SEQ ID NO:31) are provided in-Table 2.Specific examples of amino acid sequences of poxviruses of theinvention, such as Bovine popular stomatitis virus (SEQ ID NO:32),Deerpox virus (SEQ ID NO:33), lumpy skin disease virus (SEQ ID NO:34),Molluscum contagiosum virus (SEQ ID NO:35), Myxoma virus (SEQ ID NO:36),Orf virus (SEQ ID NO:37), sheeppox virus (SEQ ID NO:38), Swinepox virus(SEQ ID NO:39), Vaccinia virus (SEQ ID NO:40), Variola virus (SEQ IDNO:41), Yaba-like disease virus (SEQ ID NO:42) and Yaba-like monkeytumor virus (SEQ ID NO:43) are provided in Table 3.

Also provided herein are probes and primers for the detection of thenucleic acids of this invention and such primers and/or probes can beused alone and/or in any combination. Probes and primers of thisinvention can be prepared according to art known methods for identifyingprimer pairs and probes using art-known procedures and/or commerciallyavailable software for identifying regions that are optimal for primingand probing protocols. As would be known to one of ordinary skill in theart, any part of the A35R nucleotide sequence of this invention (or itscomplement) could be used as a primer and/or probe. A nonlimitingexample of a primer pair is: 5′ atggacgccgcgtttgttatta 3′ (SEQ ID NO:5)and 5′ tgataaaaaattactatt 3′ (SEQ ID NO:6), which could be used toamplify an A35R sequence by PCR. An example of a probe to detect A35Rnucleic acid could be 5′ tgataaaaaattactattgc 3′ (SEQ ID NO:7).—

In certain embodiments, the fragments and/or polypeptides of thisinvention can be fused with a “carrier” protein or peptide to produce afusion protein. Such fusion can be carried out, for example, by linkinga nucleic acid of this invention in frame with a nucleic acid encoding acarrier protein or fragment thereof of this invention and expressing thelinked nucleotide sequence to produce the fusion protein. For example,the carrier protein or peptide can be fused to a polypeptide and/orfragment of this invention to increase the stability thereof (e.g.,decrease the turnover rate) in the cell and/or subject. Exemplarycarrier proteins include, but are not limited to,glutathione-S-transferase or maltose-binding protein. The carrierprotein or peptide can alternatively be a reporter protein. For example,the fusion protein can comprise a polypeptide and/or fragment of thisinvention and a reporter protein or peptide (e.g., green fluorescenceprotein (GFP), β-glucoronidase, β-galactosidase, luciferase, and thelike) for easy detection of transformed cells and transgene expression.Any suitable carrier protein and/or nucleic acid encoding the carrierprotein, as is well known in the art can be used to produce a fusionprotein of this invention.

A variety of protocols for detecting the presence of and/or measuringthe amount of polypeptides, fragments and/or peptides of this inventionin a sample, using polyclonal and/or monoclonal antibodies specific forthe polypeptide, fragment and/or peptide are known in the art. Examplesof such protocols include, but are not limited to, enzyme immunoassays(EIA), agglutination assays, immunoblots (Western blot; dot/slot blot,etc.), radioimmunoassays (RIA), immunodiffusion assays,chemiluminescence assays, antibody library screens, expression arrays,enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA),immunoprecipitation, Western blotting, competitive binding assays,immunofluorescence, immunohistochemical stainingprecipitation/flocculation assays and fluorescence-activated cellsorting (FACS). These and other assays are described, among otherplaces, in Hampton et al. (Serological Methods, a Laboratory Manual, APSPress, St Paul, Minn. (1990)) and Maddox et al. (J. Exp. Med.158:1211-1216 (1993)).

Furthermore, a number of assays for identification, detection and/oramplification of nucleic acid sequences are well known in the art. Forexample, various protocols can be employed in the methods of thisinvention to amplify nucleic acid. As used herein, the term“oligonucleotide-directed amplification procedure” refers totemplate-dependent processes that result in an increase in theconcentration of a specific nucleic acid molecule relative to itsinitial concentration, or in an increase in the concentration of adetectable signal, such as amplification. As used herein, the term“oligonucleotide directed mutagenesis procedure” is intended to refer toa process that involves the template-dependent extension of a primermolecule. The term “template dependent process” refers to nucleic acidsynthesis of a RNA or a DNA molecule wherein the sequence of the newlysynthesized strand of nucleic acid is dictated by the well-known rulesof complementary base pairing. Typically, vector mediated methodologiesinvolve the introduction of the nucleic acid fragment into a DNA or RNAvector, the clonal amplification of the vector, and the recovery of theamplified nucleic acid fragment. Examples of such methodologies areprovided in U.S. Pat. No. 4,237,224 (incorporated herein by reference inits entirety). Nucleic acids, used as a template for amplificationmethods can be isolated from cells according to standard methodologies(Sambrook et al., 1989). The nucleic acid can be genomic DNA orfractionated or whole cell RNA. Where RNA is used, it may be desired toconvert the RNA to a complementary DNA. In one embodiment, the RNA canbe whole cell RNA and is used directly as the template foramplification.

Pairs of primers that selectively hybridize to nucleic acidscorresponding to the A35R gene or coding sequence are contacted with thenucleic acid under conditions that permit selective hybridization. Theterm “primer,” as defined herein, is meant to encompass any nucleic acidthat is capable of priming the synthesis of a nascent nucleic acid in atemplate dependent process. Typically, primers are oligonucleotides fromten to twenty bases in length, but shorter (e.g., 6, 7, 8, or 9 bases)or longer (e.g., 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50bases) sequences can be employed. Primers can in double-stranded orsingle-stranded form, although the single-stranded form is commonlyused.

Once hybridized, the nucleic acid: primer hybridization complex iscontacted with one or more enzymes that facilitate template-dependentnucleic acid synthesis. Multiple rounds of amplification, also referredto as “cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

Next, the amplification product is detected. In some embodiments, thedetection can be performed by visual means. Alternatively, the detectioncan involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of incorporated radiolabelor fluorescence or chemiluminescence label or even via a system usingelectrical or thermal impulse signals (e.g., Affymax technology).

A number of template dependent processes are available to amplify thesequences present in a given template sample. One of the best-knownamplification methods is the polymerase chain reaction (referred to asPCR), which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, each incorporated herein by reference in itsentirety.

Briefly, in PCR, two primer sequences are prepared that arecomplementary to regions on opposite complementary strands of the targetsequence. An excess of deoxynucleoside triphosphates is added to areaction mixture along with a DNA polymerase, e.g., a Taq polymerase. Ifthe particular target sequence is present in a sample, the primers willbind to the target sequence and the polymerase will cause the primers tobe extended along the sequence by adding on nucleotides. By raising andlowering the temperature of the reaction mixture, the extended primerswill dissociate from the target sequence to form reaction products,excess primers will bind to the target sequence and to the reactionproducts and the process is repeated.

A reverse transcriptase PCR amplification procedure can be performed inorder to quantify the amount of mRNA amplified. Methods of reversetranscribing RNA into cDNA are well known in the art (e.g., Sambrook etal., 1989). Alternative methods for reverse transcription employthermostable, RNA-dependent DNA polymerases. These methods aredescribed, for example, in PCT Publication No. WO 90/07641, filed Dec.21, 1990, incorporated herein by reference in its entirety. Polymerasechain reaction methodologies are well known in the art.

Another method for nucleic acid amplification is the ligase chainreaction (“LCR”), disclosed in Eur. Pat. Appl. No. 320308, incorporatedherein by reference in its entirety. In LCR, two complementary probepairs are prepared and in the presence of the target sequence, each pairwill bind to opposite complementary strands of the target such that theyabut. In the presence of a ligase, the two probe pairs will link to forma single unit. By temperature cycling, as in PCR, bound ligated unitsdissociate from the target and then serve as “target sequences” forligation of excess probe pairs. U.S. Pat. No. 4,883,750 (incorporated byreference herein in its entirety) describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta replicase (QβR), described in PCT Application No. PCT/US87/00880,(incorporated herein by reference), can also be used as an amplificationmethod in the present invention. In this method, a replicative sequenceof RNA that has a region complementary to that of a target is added to asample in the presence of an RNA polymerase. The polymerase will copythe replicative sequence that can then be detected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA), described in U.S. Pat. Nos.5,455,166, 5,648,211, 5,712,124 and 5,744,311, each incorporated hereinby reference, is another method of carrying out isothermal amplificationof nucleic acids which involves multiple rounds of strand displacementand synthesis, i.e., nick translation. A similar method, called RepairChain Reaction (RCR), involves annealing several probes throughout aregion targeted for amplification, followed by a repair reaction inwhich only two of the four bases are present.

The other two bases can be added as biotinylated derivatives for easydetection. A similar approach is used in SDA. Target specific sequencescan also be detected using a cyclic probe reaction (CPR). In CPR, aprobe having 3′ and 5′ sequences of non-specific DNA and a middlesequence of specific RNA is hybridized to DNA that is present in asample. Upon hybridization, the reaction is treated with RNase H, andthe products of the probe identified as distinctive products that arereleased after digestion. The original template is annealed to anothercycling probe and the reaction is repeated.

Still another amplification method, as described in Intl. Pat. Appl. No.PCT/US89/01025, which is incorporated herein by reference in itsentirety, may be used in accordance with the present invention. In oneembodiment, “modified” primers are used in a PCR-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detectable moiety (e.g.,enzyme). In another embodiment, an excess of labeled probes is added toa sample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact, available to be bound by excess probe. Cleavage of the labeledprobe signals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR (PCT Publication No. WO 88/10315,incorporated herein by reference). In NASBA, the nucleic acids can beprepared for amplification by standard phenol/chloroform extraction,heat denaturation of a clinical sample, treatment with lysis buffer andminispin columns for isolation of DNA and RNA or guanidinium chlorideextraction of RNA. These amplification techniques involve annealing aprimer that has target specific sequences. Following polymerization,DNA/RNA hybrids are digested with RNase H while double stranded DNAmolecules are heat denatured again. In either case the single strandedDNA is made fully double stranded by addition of second target specificprimer, followed by polymerization. The double-stranded DNA moleculesare then multiply transcribed by an RNA polymerase such as T7, T3 orSP6. In an isothermal cyclic reaction, the RNAs are reverse transcribedinto single stranded DNA, which is then converted to double-strandedDNA, and then transcribed once again with an RNA polymerase such as T7,T3 or SP6. The resulting products, whether truncated or complete,indicate target specific sequences.

Eur. Pat. Appl. No. 329822 (incorporated herein by reference in itsentirety) discloses a nucleic acid amplification process involvingcyclically synthesizing single stranded RNA (ssRNA), ssDNA, anddouble-stranded DNA (dsDNA), which can be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA).

The resultant ssDNA is a template for a second primer, which alsoincludes the sequences of an RNA polymerase promoter (exemplified by T7RNA polymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large Klenow fragment ofE. coli DNA polymerase I), resulting in a double-stranded DNA (dsDNA)molecule, having a sequence identical to that of the original RNAbetween the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle, leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

PCT Application WO 89/06700 (incorporated herein by reference in itsentirety) discloses a nucleic acid sequence amplification scheme basedon the hybridization of a promoter/primer sequence to a targetsingle-stranded DNA (ssDNA), followed by transcription of many RNAcopies of the sequence. This scheme is not cyclic, i.e., new templatesare not produced from the resultant RNA transcripts. Other amplificationmethods include “RACE” and “one-sided PCR” (Frohman, 1990, incorporatedby reference herein).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide,” thereby amplifying the dioligonucleotide, can alsobe used in the amplification step of the present invention.

Following any amplification, it is desirable to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products can be separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods (e.g., Sambrook et al., 1989).

Alternatively, chromatographic techniques can be used to effectseparation. There are many kinds of chromatography that can be used inthe present invention: such as, for example, adsorption, partition, ionexchange and molecular sieve, as well as many specialized techniques forusing them, including column, paper, thin-layer and gas chromatography.

Amplification products must be visualized in order to confirmamplification of the target sequences. One typical visualization methodinvolves staining of a gel with ethidium bromide and visualization underUV light. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theamplification products can then be exposed to x-ray film or visualizedunder the appropriate stimulating spectra, following separation.

In some embodiments, visualization is achieved indirectly. Followingseparation of amplification products, a labeled, nucleic acid probe isbrought into contact with the amplified target sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In other embodiments, detection can be by Southern blotting andhybridization with a labeled probe. The techniques involved in Southernblotting are well known to those of skill in the art and can be found inmany standard books on molecular protocols (e.g., Sambrook et al.,1989). Briefly, amplification products are separated by gelelectrophoresis. The gel is then contacted with a membrane, such asnitrocellulose, permitting transfer of the nucleic acid and noncovalentbinding. Subsequently, the membrane is incubated with achromophore-conjugated probe that is capable of hybridizing with atarget amplification product. Detection is by exposure of the membraneto x-ray film or ion-emitting detection devices. One example of theforegoing is described in U.S. Pat. No. 5,279,721, incorporated byreference herein, which discloses an apparatus and method for theautomated electrophoresis and transfer of nucleic acids. The apparatuspermits electrophoresis and blotting without external manipulation ofthe gel.

Additionally, a wide variety of labeling and conjugation techniques areknown in the art that are used in various nucleic acid detection andamplification assays. Methods for producing labeled hybridization probesand/or PCR or other ligation primers for detecting and/or amplifyingnucleic acid sequences can include, for example, oligolabeling, nicktranslation and end-labeling, as well as other well known methods.Alternatively, nucleic acid sequences encoding the polypeptides of thisinvention, and/or any functional fragment thereof, can be cloned into aplasmid or vector for detection and amplification. Such plasmids andvectors are well known in the art and are commercially available. It isalso contemplated that the methods of this invention can be conductedusing a variety of commercially available kits (e.g., Pharmacia &Upjohn; Promega; U.S. Biochemical Corp.). Suitable reporter molecules orlabels, which can be used for ease of detection, include, for example,radionuclides, enzymes, fluorescence agents, chemiluminescence agentsand chromogenic agents, as well as substrates, cofactors, inhibitors,magnetic particles and the like as are well known in the art.

The present invention further includes isolated polypeptides, peptides,proteins, fragments, domains and/or nucleic acid molecules that aresubstantially equivalent to those described for this invention. As usedherein, “substantially equivalent” can refer both to nucleic acid andamino acid sequences, for example a mutant sequence, that varies from areference sequence by one or more substitutions, deletions, oradditions, the net effect of which does not result in an undesirableadverse functional dissimilarity between reference and subjectsequences. In some embodiments, this invention can include substantiallyequivalent sequences that have an adverse functional dissimilarity. Forpurposes of the present invention, sequences having equivalentbiological activity and equivalent expression characteristics areconsidered substantially equivalent.

The invention further provides homologs, as well as methods of obtaininghomologs, of the polypeptides and/or fragments of this invention fromother poxviruses. As used herein, an amino acid sequence or protein isdefined as a homolog of a polypeptide or fragment of the presentinvention if it shares significant homology to one of the polypeptidesand/or fragments of the present invention. Significant homology means atleast 30%, 40%, 50%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 98% and/or 100%homology with another amino acid sequence. Specifically, by using thenucleic acids disclosed herein as a probe or as primers, and techniquessuch as PCR amplification and colony/plaque hybridization, one skilledin the art can identify homologs of the polypeptides and/or fragments ofthis invention in any mammalian-tropic poxvirus

It is further contemplated that the present invention provides kits fordetection of the polypeptides and/or fragments and/or antibodies of thisinvention in a sample. In one embodiment, the kit can comprise one ormore antibodies of this invention, along with suitable buffers, washsolutions and/or other reagents for the detection of antibody/antigencomplex formation. In an alternative embodiment, a kit of this inventioncan comprise a polypeptide, an antigenic peptide of the polypeptide ofthis invention, a fragment of this invention and/or an antigenic peptideof a fragment of this invention, along with suitable buffers, washsolutions and/or other reagents for the detection of antibody/antigencomplex formation.

The present invention further provides a kit for the detection ofnucleic acid encoding the polypeptides and/or fragments of thisinvention. For example, in one embodiment, the kit can comprise one ormore nucleic acids of this invention, along with suitable buffers, washsolutions and/or other reagents for the detection of hybridizationcomplex formation and/or amplification product formation.

It would be well understood by one of ordinary skill in the art that thekits of this invention can comprise one or more containers and/orreceptacles to hold the reagents (e.g., antibodies, antigens, nucleicacids) of the kit, along with appropriate buffers and/or wash solutionsand directions for using the kit, as would be well known in the art.Such kits can further comprise adjuvants and/or other immunostimulatoryor immunomodulating agents, as are well known in the art.

In further embodiments, the nucleic acids encoding the polypeptidesand/or fragments of this invention can be part of a recombinant nucleicacid construct comprising any combination of restriction sites and/orfunctional elements as are well known in the art that facilitatemolecular cloning and other recombinant DNA manipulations. Thus, thepresent invention further provides a recombinant nucleic acid constructcomprising a nucleic acid encoding a polypeptide and/or biologicallyactive fragment of this invention.

The present invention further provides a vector comprising a nucleicacid encoding a polypeptide and/or fragment of this invention. Thevector can be an expression vector which contains all of the geneticcomponents required for expression of the nucleic acid in cells intowhich the vector has been introduced, as are well known in the art. Theexpression vector can be a commercial expression vector or it can beconstructed in the laboratory according to standard molecular biologyprotocols. The expression vector can comprise viral nucleic acidincluding, but not limited to, poxvirus, vaccinia virus, adenovirus,retrovirus and/or adeno-associated virus nucleic acid. The nucleic acidor vector of this invention can also be in a liposome or a deliveryvehicle, which can be taken up by a cell via receptor-mediated or othertype of endocytosis.

The nucleic acid of this invention can be in a cell, which can be a cellexpressing the nucleic acid whereby a polypeptide and/or biologicallyactive fragment of this invention is produced in the cell. In addition,the vector of this invention can be in a cell, which can be a cellexpressing the nucleic acid of the vector whereby a polypeptide and/orbiologically active fragment of this invention is produced in the cell.It is also contemplated that the nucleic acids and/or vectors of thisinvention can be present in a host animal (e.g., a transgenic animal),which expresses the nucleic acids of this invention and produces thepolypeptides and/or fragments of this invention.

The nucleic acid encoding the polypeptide and/or fragment of thisinvention can be any nucleic acid that functionally encodes thepolypeptides and/or fragments of this invention. To functionally encodethe polypeptides and/or fragments (i.e., allow the nucleic acids to beexpressed), the nucleic acid of this invention can include, for example,expression control sequences, such as an origin of replication, apromoter, an enhancer and necessary information processing sites, suchas ribosome binding sites, RNA splice sites, polyadenylation sites andtranscriptional terminator sequences.

Nonlimiting examples of expression control sequences that can be presentin a nucleic acid of this invention include promoters derived frommetallothionine genes, actin genes, immunoglobulin genes, CMV, SV40,adenovirus, bovine papilloma virus, etc. A nucleic acid encoding aselected polypeptide and/or fragment can readily be determined basedupon the genetic code for the amino acid sequence of the selectedpolypeptide and/or fragment and many nucleic acids will encode anyselected polypeptide and/or fragment. Modifications in the nucleic acidsequence encoding the polypeptide and/or fragment are also contemplated.Modifications that can be useful are modifications to the sequencescontrolling expression of the polypeptide and/or fragment to makeproduction of the polypeptide and/or fragment inducible or repressibleas controlled by the appropriate inducer or repressor. Such methods arestandard in the art. The nucleic acid of this invention can be generatedby means standard in the art, such as by recombinant nucleic acidtechniques and/or by synthetic nucleic acid synthesis or in vitroenzymatic synthesis.

The nucleic acids and/or vectors of this invention can be transferredinto a host cell (e.g., a prokaryotic or eukaryotic cell) by well-knownmethods, which vary depending on the type of cell host. For example,calcium chloride transfection is commonly used for prokaryotic cells,whereas calcium phosphate treatment, transduction and/or electroporationcan be used for other cell hosts.

The present invention also provides various screening assays to identifysubstances that either enhance or inhibit the activity of A35R, eitherat the protein or nucleic acid level.

In one embodiment, provided herein is a method of identifying asubstance having the ability to inhibit or enhance the binding activityof a polypeptide and/or biologically active fragment of this inventioncomprising contacting the substance with the A35R protein or abiologically active fragment thereof under conditions whereby bindingcan occur and detecting a decrease or increase in the amount of bindingin the presence of the substance as compared to a control amount ofbinding in the absence of the substance, thereby identifying a substancehaving the ability to inhibit or enhance the binding activity of theA35R protein.

Inhibition or enhancement of binding activity can be detected by any ofa variety of art-recognized methods for evaluating binding activity. Asone example, the substance to be tested and the A35R polypeptide and/orfragment can be contacted in the presence of target cells or a targetsubstrate known to bind the A35R polypeptide or fragment. The amount ofbinding of polypeptide or fragment to the cells or the substrate in thepresence of the substance and the amount of binding of polypeptide orfragment to the cells or the substrate in the absence of the substanceis determined and a decrease or increase in the amount of binding in thepresence of the substance identifies the substance as having the abilityto inhibit or enhance binding. Nonlimiting examples of binding targetsfor A35R protein include antibodies, antibody fragments, ligands,cellular proteins, viral proteins, small molecules and/or drugs thatspecifically bind A35R protein or an active fragment thereof.

In some embodiments, binding of the A35R polypeptide and/or fragment totarget cells or a target substrate can be measured by attaching adetectable moiety to the polypeptide or fragment (e.g., a fluorescencemoiety, histochemically detectable moiety, radioactive moiety, etc.).The amount of detectable moiety can be measured in the presence andabsence of the substance to be tested and the amounts can be compared todetermine inhibition or enhancement.

In addition, the present invention provides a method of identifying asubstance having the ability to inhibit or enhance the immunomodulatingactivity of an A35R polypeptide and/or a biologically active fragment ofthis invention, comprising contacting the substance with the A35Rpolypeptide of this invention and/or a biologically active fragmentthereof under conditions whereby immunomodulating activity can occur anddetecting a decrease or increase in the amount of immunomodulatingactivity in the presence of the substance as compared to a controlamount of immunomodulating activity in the absence of the substance,thereby identifying a substance having the ability to inhibit or enhancethe immunomodulating activity of the A35R protein.

Inhibition or enhancement of the immunomodulating activity of the A35Rprotein can be detected by any of a variety of art-recognized methodsfor evaluating immunomodulating activity, including the assays fornitric oxide production and interleukin 2 (IL-2) secretion as describedin the Examples section herein.

Further provided is a method of identifying a substance having theability to enhance or inhibit the immunogenic activity of the A35Rprotein of this invention and/or a biologically active fragment thereof,comprising contacting the substance with the A35R protein or animmunogenic fragment thereof under conditions whereby a measurableimmune response can be elicited and detecting an increase or decrease inthe amount of immune response in the presence of the substance, ascompared to a control amount of immune response in the absence of thesubstance, thereby identifying a substance having the ability to enhanceor inhibit immunogenic activity of the A35R protein. Assays to detectand measure immune responses are well known in the art and can beemployed to detect either humoral or cellular immune responses.

It is also contemplated that the present invention includes methods ofscreening poxviruses for mutants defective in one or more of thebiological activities of the A35R protein, for use, for example, in avaccine preparation. Such mutants can be identified as having a defectin any of the biological activities of the A35R protein according to theprotocols described herein and as are known in the art. Such mutants canbe further tested for being attenuated in the ability to produce aclinical infection in a subject (i.e., for virulence potential) and thenfurther evaluated for use as a vaccine according to known protocols.

For example, in one embodiment, poxviruses containing a mutation in theA35R coding sequence or lacking the A35R coding sequence) can begenerated through such art-known techniques as gene disruption and theirvirulence potential determined by challenge studies in animals and byassessments of immunomodulating activity as described herein. Inaddition, complementation studies can be performed to restore thedefective activity of the A35R protein, in order to characterize themutant.

DEFINITIONS

As used herein, “a” or “an” or “the” can mean one or more than one. Forexample, “a” cell can mean one cell or a plurality of cells.

Also as used herein, “and/or” refers to and encompasses any and allpossible combinations of one or more of the associated listed items, aswell as the lack of combinations when interpreted in the alternative(“or”).

Furthermore, the term “about,” as used herein when referring to ameasurable value such as an amount of a compound or agent of thisinvention, dose, time, temperature, and the like, is meant to encompassvariations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of thespecified amount.

A poxvirus of this invention includes but is not limited to vacciniavirus, molluscum contagiosum, camelpox virus, cowpox virus, ectromelia(mousepox) virus, rabbitpox virus, monkeypox virus, raccoonpox virus,taterapox virus, buffalopox virus variola major virus, variola minorvirus, lumpy skin disease virus, swinepox virus, Yaba virus, sheeppoxvirus, myxoma virus volepox virus and any other poxvirus that has anA35R protein, either now known or later identified. Additionalpoxviruses are listed in Table 1.

The A35R gene and A35R protein of this invention is an A35R gene or A35Rprotein or its ortholog from a poxvirus of this invention. The A35R geneencodes a 176 amino acid protein in VV Copenhagen strain, modifiedvaccinia Ankara strain and Tian Tan strain (GenBank AF095689). TheVV-A35R orthologs are conserved in all poxviruses with a mammalian hostrange, ranging in size from 176 to 192 amino acids in all sequencedviruses, except for a large ortholog of 233 amino acids in molluscumcontagiosum virus. The gene is fragmented into 60 amino acid and 50amino acid open reading frames (ORFs) in variola virus sequences (India,Bangladesh and Garcia).

As used herein, “modulate,” “modulates” or “modulation” refers toenhancement (e.g., an increase) or inhibition (e.g., diminished, reducedor suppressed) of the specified activity.

The term “enhancement,” “enhance,” “enhances,” or “enhancing” refers toan increase in the specified parameter (e.g., at least about a 1.1-fold,1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold,10-fold, twelve-fold, or even fifteen-fold or more increase) and/or anincrease in the specified activity of at least about 5%, 10%, 25%, 35%,40%, 50%, 60%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%.

The term “inhibit,” “diminish,” “reduce” or “suppress” refers to adecrease in the specified parameter (e.g., at least about a 1.1-fold,1.25-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 8-fold,10-fold, twelve-fold, or even fifteen-fold or more increase) and/or adecrease or reduction in the specified activity of at least about 5%,10%, 25%, 35%, 40%, 50%, 60%, 75%, 80%, 90%, 95%, 97%, 98%, 99% or 100%.In particular embodiments, the inhibition or reduction results in littleor essentially no detectible activity (at most, an insignificant amount,e.g., less than about 10% or about 5%).

As used herein, the transitional phrase “consisting essentially of”means that the scope of a claim is to be interpreted to encompass thespecified materials or steps recited in the claim, “and those that donot materially affect the basic and novel characteristic(s)” of theclaimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461,463 (CCPA 1976) (emphasis in the original); see also MPEP §2111.03.Thus, the term “consisting essentially of” when used in a claim of thisinvention is not intended to be interpreted to be equivalent to“comprising.”

“Isolated” as used herein means the nucleic acid or protein or proteinfragment of this invention is sufficiently free of contaminants or cellcomponents with which nucleic acids or proteins normally occur.“Isolated” does not mean that the preparation is technically pure(homogeneous), but it is sufficiently pure to provide the nucleic acidor protein or protein fragment in a form in which it can be usedtherapeutically.

“Epitope” or “antigenic epitope” or “antigenic peptide” as used hereinmeans a specific amino acid sequence which, when present in the properconformation, provides a reactive site for an antibody or T cellreceptor. The identification of epitopes on antigens can be carried outby immunology protocols that are well known in the art. Typically, anepitope or antigenic peptide can be 4, 5, 6, 7, 8, 9, 10, 12, 14, 16,18, 20, 25, 30, 35, 40, 45 or 50 amino acids in length.

As used herein, the term “polypeptide” or “protein” is used to describea chain of amino acids that correspond to those encoded by a nucleicacid. A polypeptide of this invention can be a peptide, which usuallydescribes a chain of amino acids of from two to about 30 amino acids.The term polypeptide as used herein also describes a chain of aminoacids having more than 30 amino acids and can be a fragment or domain ofa protein or a full length protein. Furthermore, as used herein, theterm polypeptide can refer to a linear chain of amino acids or it canrefer to a chain of amino acids that has been processed and folded intoa functional protein. It is understood, however, that 30 is an arbitrarynumber with regard to distinguishing peptides and polypeptides and theterms can be used interchangeably for a chain of amino acids. Thepolypeptides of the present invention are obtained by isolation andpurification of the polypeptides from cells where they are producednaturally, by enzymatic (e.g., proteolytic) cleavage, and/orrecombinantly by expression of nucleic acid encoding the polypeptides orfragments of this invention. The polypeptides and/or fragments of thisinvention can also be obtained by chemical synthesis or other knownprotocols for producing polypeptides and fragments.

The amino acid sequences disclosed herein are presented in the amino tocarboxy direction, from left to right. Nucleotide sequences arepresented herein in the 5′ to 3′ direction, from left to right. It isintended that the nucleic acids of this invention can be either singleor double stranded (i.e., including the complementary nucleic acid). Anucleic acid of this invention can be the complement of a nucleic aciddescribed herein.

A “biologically active fragment” or “active fragment” as used hereinincludes a polypeptide of this invention that comprises a sufficientnumber of amino acids to have one or more of the biological activitiesof the polypeptides of this invention. Such biological activities caninclude, but are not limited to, in any combination, binding activity,immunomodulating activity, virulence activity and/or immunogenicactivity, as well as any other activity now known or later identifiedfor the polypeptides and/or fragments of this invention. A fragment of apolypeptide of this invention can be produced by methods well known androutine in the art. Fragments of this invention can be produced, forexample, by enzymatic or other cleavage of naturally occurring peptidesor polypeptides or by synthetic protocols that are well known. Suchfragments can be tested for one or more of the biological activities ofthis invention according to the methods described herein, which areroutine methods for testing activities of polypeptides, and/or accordingto any art-known and routine methods for identifying such activities.Such production and testing to identify biologically active fragments ofthe polypeptides described herein would be well within the scope of oneof ordinary skill in the art and would be routine.

Fragments of the polypeptides of this invention are preferably at leastabout ten amino acids in length and retain one or more of the biologicalactivities (e.g., immunomodulating; virulence activities) and/or theimmunological activities of the A35R protein. Examples of the fragmentsof this invention include, but are not intended to be limited to, thefollowing fragments identified by the amino acid number as shown in theSequence Listing for SEQ ID NO:2: Amino acids 1-10, 10-20, 20-30, 30-40,40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 110-120, 120-130, 130-140,140-150, 150-160, 160-170, 170-180, 180-190, 190-200, 200-210, 210-220,220-230, 230-240, 240-250, 1-25, 1-50, 1-67, 1-75, 1-100, 1-125, 1-135,1-145, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-250, 68-180, 183-223,50-100, 100-200, 200-250, etc.

It is understood that this list is exemplary only and that a fragment ofthis invention can be any amino acid sequence containing any combinationof contiguous amino acids that are numbered in the Sequence Listing asamino acids 1 through 250 even if that combination is not specificallyrecited as an example herein. It is also understood that these fragmentscan be combined in any order or amount. For example, fragment 1-10 canbe combined with fragment 10-20 to produce a fragment of amino acids1-20. As another example, fragment 1-20 can be combined with fragment50-60 to produce a single fragment of this invention having 31 aminoacids (AA 10-20 and AA 50-60). Also fragments can be present in multiplenumbers and in any combination in a fragment of this invention. Thus,for example, fragment 1-150 can be combined with a second fragment 1-150and/or combined with fragment 225-230 to produce a fragment of thisinvention. Other exemplary fragments of this invention include aminoacids 1-60 of variola virus; and the amino acid sequence:YDDLDISIMDFIGPY (SEQ ID NO:44).

The terms “homology,” “identity” and “complementarity” as used hereinrefer to a degree of similarity between two or more sequences. There maybe partial homology or complete homology (i.e., identity). A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as“substantially homologous,” The inhibition of hybridization of thecompletely complementary sequence to the target sequence can be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or hybridization probe will competefor and inhibit the binding of a completely homologous sequence to thetarget sequence under conditions of low stringency, as this term isknown in the art. This is not to say that conditions of low stringencyare such that non-specific binding is permitted; low stringencyconditions require that the binding of two sequences to one another be aspecific (i.e., selective) interaction. The absence of non-specificbinding can be tested by the use of a second target sequence that lackseven a partial degree of complementarity (e.g., less than about 30%identity). In the absence of non-specific binding, the probe will nothybridize to the second non-complementary target sequence.

The term “hybridization” as used herein refers to any process by which afirst strand of nucleic acid binds with a second strand of nucleic acidthrough base pairing. Nucleic acids encoding the polypeptides and/orfragments of this invention can be detected by DNA-DNA or DNA-RNAhybridization and/or amplification using probes, primers and/orfragments of polynucleotides encoding the polypeptides and/or fragmentsof this invention and/or designed to detect and/or amplify the nucleicacids of this invention.

The term “hybridization complex” as used herein refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,paper, membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells and/or nucleic acids have beenfixed).

The term “nucleotide sequence” refers to a heteropolymer of nucleotidesor the sequence of these nucleotides. The terms “nucleic acid,”“oligonucleotide” and “polynucleotide” are also used interchangeablyherein to refer to a heteropolymer of nucleotides. Generally, nucleicacid segments provided by this invention may be assembled from fragmentsof the genome and short oligonucleotide linkers, or from a series ofoligonucleotides, or from individual nucleotides, to provide a syntheticnucleic acid which is capable of being expressed in a recombinanttranscriptional unit comprising regulatory elements derived from amicrobial or viral operon, or a eukaryotic gene. Nucleic acids of thisinvention can comprise a nucleotide sequence that can be identical insequence to the sequence which is naturally occurring or, due to thewell-characterized degeneracy of the nucleic acid code, can includealternative codons that encode the same amino acid as that which isfound in the naturally occurring sequence. Furthermore, nucleic acids ofthis invention can comprise nucleotide sequences that can include codonswhich represent conservative substitutions of amino acids as are wellknown in the art, such that the biological activity of the resultingpolypeptide and/or fragment is retained.

The term “probe” or “primer” includes naturally occurring and/orrecombinant and/or chemically synthesized single- and/or double-strandednucleic acids. They can be labeled for detection by nick translation,Klenow fill-in reaction, PCR and/or other methods well known in the art.Probes and primers of the present invention, their preparation and/orlabeling are described in Sambrook et al. 1989. Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, NY and Ausubel et al.1989. Current Protocols in Molecular Biology, John Wiley & Sons, NewYork N.Y., both of which are incorporated herein by reference in theirentirety for these teachings.

The term “stringent” as used herein refers to hybridization conditionsthat are commonly understood in the art to define the conditions of thehybridization procedure. Stringency conditions can be low, high ormedium, as those terms are commonly know in the art and well recognizedby one of ordinary skill. In various embodiments, stringent conditionscan include, for example, highly stringent (i.e., high stringency)conditions (e.g., hybridization in 0.5 M NaHPO₄, 7% sodium dodecylsulfate (SDS), 1 mM EDTA at 65° C., and washing in 0.1×SSC/0.1% SDS at68° C.), and/or moderately stringent (i.e., medium stringency)conditions (e.g., washing in 0.2×SSC/0.1% SDS at 42° C.).

“Amplification” as used herein includes the production of multiplecopies of a nucleic acid molecule and is generally carried out usingpolymerase chain reaction (PCR) and/or any other amplificationtechnologies as are well known in the art (Dieffenbach and Dveksler.1995. PCR Primer, a Laboratory Manual, Cold Spring Harbor Press,Plainview, N.Y.).

The term “sample” as used herein is used in its broadest sense. Abiological sample suspected of containing a polypeptide, fragment,antibody and/or nucleic acid of this invention can be any biologicalfluid, an extract from a cell, an extracellular matrix isolated from acell, a cell (in solution or bound to a solid support), a tissue, atissue print, and the like. A sample of this invention can also includea substance not obtained from the body of a subject of this invention.Examples of such a sample include but are not limited to, a water orfluid sample, a food or foodstuff sample, a plant or plant materialsample, a soil or rock sample, an animal or animal material sample, ananimal bedding sample, an animal cage sample, air sample a soil or dirtsample, a cloth, paper or other material used to swab, wipe, dust orclean a surface, an effluent sample, etc.

“Effective amount” as used herein refers to an amount of a compound,agent, substance or composition of this invention that is sufficient toproduce a desired effect, which can be a therapeutic effect. Theeffective amount will vary with the age, general condition of thesubject, the severity of the condition being treated, the particularcompound, agent, substance or composition administered, the duration ofthe treatment, the nature of any concurrent treatment, thepharmaceutically acceptable carrier used if any, and like factors withinthe knowledge and expertise of those skilled in the art. As appropriate,an “effective amount” in any individual case can be determined by one ofordinary skill in the art by reference to the pertinent texts andliterature and/or by using routine experimentation. (Remington, TheScience And Practice of Pharmacy (20th ed. 2000)).

A “pharmaceutically acceptable” component such as a salt, carrier,excipient or diluent of a composition according to the present inventionis a component that (i) is compatible with the other ingredients of thecomposition in that it can be combined with the compositions of thepresent invention without rendering the composition unsuitable for itsintended purpose, and (ii) is suitable for use with subjects as providedherein without undue adverse side effects (such as toxicity, irritation,and allergic response). Side effects are “undue” when their riskoutweighs the benefit provided by the composition. Non-limiting examplesof pharmaceutically acceptable components (e.g., pharmaceuticallyacceptable carriers) include, without limitation, any of the standardpharmaceutical carriers such as phosphate buffered saline solutions,water, emulsions such as oil/water emulsion, microemulsions and varioustypes of wetting agents. In particular, it is intended that apharmaceutically acceptable carrier be a sterile carrier that isformulated for administration to or delivery into a subject of thisinvention.

Furthermore, any of the compositions of this invention can comprise apharmaceutically acceptable carrier and a suitable adjuvant. As usedherein, “suitable adjuvant” describes an adjuvant capable of beingcombined with the polypeptide and/or fragment and/or nucleic acid ofthis invention to further enhance an immune response without deleteriouseffect on the subject or the cell of the subject. A suitable adjuvantcan be, but is not limited to, MONTANIDE ISA51 (Seppic, Inc., Fairfield,N.J.), SYNTEX adjuvant formulation 1 (SAF-1), composed of 5 percent(wt/vol) squalene (DASF, Parsippany, N.J.), 2.5 percent Pluronic, L121polymer (Aldrich Chemical, Milwaukee), and 0.2 percent polysorbate(Tween 80, Sigma) in phosphate-buffered saline. Other suitable adjuvantsare well known in the art and include QS-21, Freund's adjuvant (completeand incomplete), alum, aluminum phosphate, aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE) and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trealosedimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalene/Tween 80emulsion.

The compositions of the present invention can also include othermedicinal agents, pharmaceutical agents, carriers, diluents,immunostimulatory cytokines, etc. Actual methods of preparing suchdosage forms are known, or will be apparent, to those skilled in thisart.

A “tumor antigen” of this invention (i.e., an antigen specificallyassociated with tumor cells) of this invention can include, for example,HER2/neu and BRCA1 antigens for breast cancer, MART-1/MelanA, gp100,tyrosinase, TRP-1, TRP-2, NY-ESO-1, CDK-4, β-catenin, MUM-1, Caspase-8,KIAA0205, HPVE7, SART-1, PRAME, and p15 antigens, members of the MAGEfamily, the BAGE family (such as BAGE-1), the DAGE/PRAME family (such asDAGE-1), the GAGE family, the RAGE family (such as RAGE-1), the SMAGEfamily, NAG, TAG-72, CA125, mutated proto-oncogenes such as p21ras,mutated tumor suppressor genes such as p53; tumor associated viralantigens (e.g., HPV16 E7), the SSX family, HOM-MEL-55, NY-COL-2,HOM-HD-397, HOM-RCC-1.14, HOM-HD-21, HOM-NSCLC-11, HOM-MEL-2.4,HOM-TES-11, RCC-3.1.3, NY-ESO-1, and the SCP family. Members of the MAGEfamily include, but are not limited to, MAGE-1, MAGE-2, MAGE-3, MAGE-4and MAGE-11. Members of the GAGE family include, but are not limited to,GAGE-1, GAGE-6, See, e.g., review by Van den Eynde and van der Bruggen(1997) in Curr. Opin. Immunol. 9: 684-693, Sahin et al. (1997) in Curr.Opin. Immunol. 9: 709-716, and Shawler et al. (1997), the entirecontents of which are incorporated by reference herein for theirteachings of cancer antigens.

The tumor antigen can also be, but is not limited to, human epithelialcell mucin (Muc-1; a 20 amino acid core repeat for Muc-1 glycoprotein,present on breast cancer cells and pancreatic cancer cells), MUC-2,MUC-3, MUC-18, the Ha-ras oncogene product, carcino-embryonic antigen(CEA), the raf oncogene product, CA-125, GD2, GD3, GM2, TF, sTn, gp75,EBV-LMP 1 & 2, HPV-F4, 6, 7, prostatic serum antigen (PSA),prostate-specific membrane antigen (PSMA), alpha-fetoprotein (AFP),CO17-1A, GA733, gp72, p53, the ras oncogene product, β-HCG, gp43,HSP-70, p17 mel, HSP-70, gp43, HMW, HOJ-1, melanoma gangliosides,TAG-72, mutated proto-oncogenes such as p21ras, mutated tumor suppressorgenes such as p53, estrogen receptor, milk fat globulin, telomerases,nuclear matrix proteins, prostatic acid phosphatase, protein MZ2-E,polymorphic epithelial mucin (PEM), folate-binding-protein LK26,truncated epidermal growth factor receptor (EGFR), Thomsen-Friedenreich(T) antigen, GM-2 and GD-2 gangliosides, polymorphic epithelial mucin,folate-binding protein LK26, human chorionic gonadotropin (HCG),pancreatic oncofetal antigen, cancer antigens 15-3, 19-9, 549, 195,squamous cell carcinoma antigen (SCCA), ovarian cancer antigen (OCA),pancreas cancer associated antigen (PaA), mutant K-ras proteins, mutantp53, and chimeric protein p210_(BCR-ABL) and tumor associated viralantigens (e.g., HPV16 E7).

The tumor antigen of this invention can also be an antibody produced bya B cell tumor (e.g., B cell lymphoma; B cell leukemia; myeloma; hairycell leukemia), a fragment of such an antibody, which contains anepitope of the idiotype of the antibody, a malignant B cell antigenreceptor, a malignant B cell immunoglobulin idiotype, a variable regionof an immunoglobulin, a hypervariable region or complementaritydetermining region (CDR) of a variable region of an immunoglobulin, amalignant T cell receptor (TCR), a variable region of a TCR and/or ahypervariable region of a TCR. In one embodiment, the cancer antigen ofthis invention can be a single chain antibody (scFv), comprising linkedV_(H), and V_(L) domains, which retains the conformation and specificbinding activity of the native idiotype of the antibody.

The present invention is in no way limited to the tumor antigens listedherein. Other tumor antigens be identified, isolated and cloned bymethods known in the art such as those disclosed in U.S. Pat. No.4,514,506, the entire contents of which are incorporated by referenceherein.

The cancer to be treated by the methods of this invention can be, but isnot limited to, B cell lymphoma, T cell lymphoma, myeloma, leukemia,hematopoietic neoplasias, thymoma, lymphoma, sarcoma, lung cancer, livercancer, non-Hodgkins lymphoma, Hodgkins lymphoma, uterine cancer,adenocarcinoma, breast cancer, pancreatic cancer, colon cancer, lungcancer, renal cancer, bladder cancer, liver cancer, prostate cancer,ovarian cancer, primary or metastatic melanoma, squamous cell carcinoma,basal cell carcinoma, brain cancer, angiosarcoma, hemangiosarcoma, headand neck carcinoma, thyroid carcinoma, soft tissue sarcoma, bonesarcoma, testicular cancer, uterine cancer, cervical cancer,gastrointestinal cancer, and any other cancer now known or lateridentified (see, e.g., Rosenberg (1996) Ann. Rev. Med. 47:481-491, theentire contents of which are incorporated by reference herein).

Infectious and/or pathogenic agents of this invention can include, butare not limited to, Hepadnaviridae, including hepatitis A, B, C, D, E,F, G, etc. (e.g., HBsAg, HBcAg, HBeAg); Flaviviridae, including humanhepatitis C virus (HCV), yellow fever virus and dengue viruses;Retroviridae, including human immunodeficiency viruses (HIV) and human Tlymphotropic viruses (HTLV1 and HTLV2); Herpesviridae, including herpessimplex viruses (HSV-1 and HSV-2), Epstein Barr virus (EBV),cytomegalovirus, varicella-zoster virus (VZV), human herpes virus 6(HHV-6) human herpes virus 8 (HHV-8), and herpes B virus; Papovaviridae,including human papilloma viruses; Rhabdoviridae, including rabiesvirus; Paramyxoviridae, including respiratory syncytial virus;Reoviridae, including rotaviruses; Bunyaviridae, including hantaviruses;Filoviridae, including Ebola virus; Adenoviridae; Parvoviridae,including parvovirus B-19; Arenaviridae, including Lassa virus;Orthomyxoviridae, including influenza viruses; Poxyiridae, including Orfvirus, molluscum contageosum virus, smallpox virus and Monkeypox virus;Togaviridae, including Venezuelan equine encephalitis virus;Coronaviridae, including corona viruses such as the severe acuterespiratory syndrome (SARS) virus; Picornaviridae includingpolioviruses; rhinoviruses; orbiviruses; picodnaviruses;encephalomyocarditis virus (EMV); parainfluenza viruses, adenoviruses,coxsackieviruses, echoviruses, rubeola virus, rubella virus, humanpapillomaviruses, canine distemper virus, canine contagious hepatitisvirus, feline calicivirus, feline rhinotracheitis virus, TGE virus(swine), foot and mouth disease virus, simian virus 5, humanparainfluenza virus type 2, human metapneuomovirus, enteroviruses, andany other pathogenic virus now known or later identified (see, e.g.,Fundamental Virology, Fields et al., Eds., 3^(rd) ed., Lippincott-Raven,New York, 1996, the entire contents of which are incorporated byreference herein for the teachings of pathogenic viruses).

A pathogenic microorganism of this invention can include but is notlimited to, Rickettsia, Chlamydia, Mycobacteria, Clostridia,Corynebacteria, Mycoplasma, Ureoaplasma, Legionella, Shigella,Salmonella, pathogenic Escherichia coli species, Bordatella, Neisseria,Treponema, Bacillus, Haemophilus, Moraxella, Vibrio, Staphylococcusspp., Streptococcus spp., Campylobacter spp., Borrelia spp., Leptospiraspp., Erlichia spp., Klebsiella spp., Pseudomonas spp., Helicobacterspp., and any other pathogenic microorganism now known or lateridentified (see, e.g., Microbiology, Davis et al, Eds., 4^(th) ed.,Lippincott, New York, 1990, the entire contents of which areincorporated herein by reference for the teachings of pathogenicmicroorganisms).

Specific examples of microorganisms include, but are not limited to,Helicobacter pylori, Chlamydia pneumoniae, Chlamydia trachomatis,Ureaplasma urealyticum, Mycoplasma pneumoniae, Staphylococcus aureus,Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcusviridans, Enterococcus faecalis, Neisseria meningitidis, Neisseriagonorrhoeae, Treponema pallidum, Bacillus anthracis, Salmonella typhi,Vibrio cholera, Pasteurella pestis, Pseudomonas aeruginosa,Campylobacter jejuni, Clostridium difficile, Clostridium botulinum,Mycobacterium tuberculosis, Borrelia burgdorferi, Haemophilus ducreyi,Corynebacterium diphtheria, Bordetella pertussis, Bordetellaparapertussis, Bordetella bronchiseptica, Haemophilus influenza, andenterotoxic Escherichia coli.

Pathogenic protozoa can include, but are not limited to, Plasmodiumspecies (e.g., malaria antigens), Babeosis species, Schistosoma species,Trypanosoma species, Pneumocystis carnii, Toxoplasma species, Leishmaniaspecies, and any other protozoan pathogen now known or later identified.

Also included are pathogenic yeast and fungi, examples of which can be,but are not limited to, Aspergillus species, Candida species,Cryptococcus species. Histoplasma species, Coccidioides species, and anyother pathogenic fungus now known or later identified.

Transplantation antigens of this invention include, but are not limitedto, different antigenic specificities of HLA-A, B and C Class Iproteins. Different antigenic specificities of HLA-DR, HLA-DQ, HLA-DPand HLA-DW Class II proteins can also be used (WHO NomenclatureCommittee, Immunogenetics 16:135 (1992); Hensen et al., in FundamentalImmunology, Paul, Ed., pp. 577-628, Raven Press, New York, 1993; NIHGenBank and EMBL data bases).

The present invention also contemplates the treatment or prevention ofallergies and/or allergic reaction, caused by various allergens, whichcan include, but are not limited to, environmental allergens such asdust mite allergens; plant allergens such as pollen, including ragweedpollen; insect allergens such as bee and ant venom; and animal allergenssuch as cat dander, dog dander and animal saliva allergens.

An “immunomodulatory molecule” of this invention can be, but is notlimited to an immunostimulatory cytokine that can be, but is not limitedto, GM/CSF, interleukin-2, interleukin-12, interferon-gamma,interleukin-4, tumor necrosis factor-alpha, interleukin-1, hematopoieticfactor flt3L, CD40L, B7.1 co-stimulatory molecules and B7.2co-stimulatory molecules.

Additional examples of an immunomodulatory molecule of this inventioninclude the adjuvants of this invention, including, for example, SYNTEXadjuvant formulation 1 (SAF-1) composed of 5 percent (wt/vol) squalene(DASF, Parsippany, N.J.), 2.5 percent Pluronic, L121 polymer (AldrichChemical, Milwaukee), and 0.2 percent polysorbate (Tween 80, Sigma) inphosphate-buffered saline. Suitable adjuvants also include an aluminumsalt such as aluminum hydroxide gel (alum), aluminum phosphate, oralgannmulin, but may also be a salt of calcium, iron or zinc, or may bean insoluble suspension of acylated tyrosine, or acylated sugars,cationically or anionically derivatized polysaccharides, orpolyphosphazenes.

Other adjuvants are well known in the art and include QS-21, Freund'sadjuvant (complete and incomplete), aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE) and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trealosedimycolate and cell wall skeleton (MPL+TDM+CWS) in 2% squalene/Tween 80emulsion.

Additional adjuvants can include, for example, a combination ofmonophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl. lipidA (3D-MPL) together with an aluminum salt. An enhanced adjuvant systeminvolves the combination of a monophosphoryl lipid A and a saponinderivative, particularly the combination of QS21 and 3D-MPL as disclosedin PCT publication number WO 94/00153 (the entire contents of which areincorporated herein by reference), or a less reactogenic compositionwhere the QS21 is quenched with cholesterol as disclosed in PCTpublication number WO 96/33739 (the entire contents of which areincorporated herein by reference). A particularly potent adjuvantformulation involving QS21 3D-MPL & tocopherol in an oil in wateremulsion is described in PCT publication number WO 95/17210 (the entirecontents of which are incorporated herein by reference). In addition,the nucleic acid of this invention can include an adjuvant by comprisinga nucleotide sequence encoding an A35R protein or active fragmentthereof of this invention and a nucleotide sequence that provides anadjuvant function, such as CpG sequences. Such CpG sequences, or motifs,are well known in the art.

The terms “treat,” “treating” or “treatment” include any type of actionthat imparts a modulating effect, which, for example, can be abeneficial effect, to a subject afflicted with a disorder, disease,condition or illness, including improvement in the disorder, disease,condition or illness of the subject (e.g., in one or more symptoms),delay in the progression of the disorder, disease, condition or illness,prevention or delay of the onset of the disorder, disease, condition orillness, and/or change in clinical parameters, disorder, disease,condition or illness status, etc., as would be well known in the art.

As used herein, the term “antibody” includes intact immunoglobulinmolecules as well as fragments thereof that are capable of binding theepitopic determinant of an antigen (i.e., antigenic determinant).Antibodies that bind the polypeptides of this invention are preparedusing intact polypeptides or fragments as the immunizing antigen. Thepolypeptide or fragment used to immunize an animal can be derived fromenzymatic cleavage, recombinant expression, isolation from biologicalmaterials, synthesis, etc., and can be conjugated to a carrier protein,if desired. Commonly used carriers that are chemically coupled topeptides and proteins for the production of antibody include, but arenot limited to, bovine serum albumin, thyroglobulin and keyhole limpethemocyanin. The coupled peptide or protein is then used to immunize theanimal (e.g., a mouse, rat, or rabbit). The polypeptide or peptideantigens can also be administered with an adjuvant, as described hereinand as otherwise known in the art.

An antibody of this invention can be any type of immunoglobulin,including IgG, IgM, IgA, IgD, and/or IgE. The antibody can be monoclonalor polyclonal and can be of any species of origin, including, forexample, mouse, rat, rabbit, horse, goat, sheep or human, or can be achimeric or humanized antibody (e.g., Walker et al., Molec. Immunol.26:403-11 (1989)). The antibodies can be recombinant monoclonalantibodies produced according to the methods disclosed in U.S. Pat. No.4,474,893 or U.S. Pat. No. 4,816,567. The antibodies can also bechemically constructed according to methods disclosed in U.S. Pat. No.4,676,980. The antibody can further be a single chain antibody (e.g.,scFv) or bispecific antibody.

Antibody fragments included within the scope of the present inventioninclude, for example, Fab, F(ab′)2, and Fc fragments, and thecorresponding fragments obtained from antibodies other than IgG. Suchfragments can be produced by known techniques. For example, F(ab′)2fragments can be produced by pepsin digestion of the antibody molecule,and Fab fragments can be generated by reducing the disulfide bridges ofthe F(ab′)2 fragments. Alternatively, Fab expression libraries can beconstructed to allow rapid and easy identification of monoclonal Fabfragments with the desired specificity (Huse et al., (1989) Science254:1275-1281). Antibodies can also be obtained by phage displaytechniques known in the art or by immunizing a heterologous host with acell containing an epitope of interest.

The polypeptide, fragment or antigenic epitope that is used as animmunogen can be modified or administered in an adjuvant in order toincrease antigenicity. Methods of increasing the antigenicity of aprotein or peptide are well known in the art and include, but are notlimited to, coupling the antigen with a heterologous protein (such asglobulin or β-galactosidase) or through the inclusion of an adjuvantduring immunization.

For example, for the production of antibodies, various hosts includinggoats, rabbits, rats, mice, humans, and others, can be immunized byinjection with the polypeptides and/or fragments of this invention, withor without a carrier protein. Additionally, various adjuvants may beused to increase the immunological response. Such adjuvants include, butare not limited to, Freund's complete and incomplete adjuvants, mineralgels such as aluminum hydroxide, and surface-active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, and dinitrophenol. Among adjuvants used inhumans, BCG (bacilli Calmette-Guerin) and Corynebacterium parvum areespecially preferable.

Monoclonal antibodies can be produced in a hybridoma cell line accordingto the technique of Kohler and Milstein (Nature 265:495-97 (1975)).Other techniques for the production of monoclonal antibodies include,but are not limited to, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kozbor et al. 1985. J. Immunol. Methods81:31-42; Cote et al. 1983. Proc. Natl. Acad. Sci. 80:2026-2030; Cole etal. 1984. Mol. Cell Biol. 62:109-120).

For example, to produce monoclonal antibodies, a solution containing theappropriate antigen can be injected into a mouse and, after a sufficienttime, the mouse sacrificed and spleen cells obtained. The spleen cellsare then immortalized by fusing them with myeloma cells or with lymphomacells, typically in the presence of polyethylene glycol, to producehybridoma cells. The hybridoma cells are then grown in a suitable mediumand the supernatant screened for monoclonal antibodies having thedesired specificity. Monoclonal Fab fragments can be produced in abacterial cell such as E. coli by recombinant techniques known to thoseskilled in the art (e.g., Huse, Science 246:1275-81 (1989)). Any one ofa number of methods well known in the art can be used to identify thehybridoma cell, which produces an antibody with the desiredcharacteristics. These include screening the hybridomas by ELISA assay,Western blot analysis, or radioimmunoassay. Hybridomas secreting thedesired antibodies are cloned and the class and subclass are identifiedusing standard procedures known in the art.

For polyclonal antibodies, antibody-containing serum is isolated fromthe immunized animal and is screened for the presence of antibodies withthe desired specificity using any of the well known procedures asdescribed herein.

The present invention further provides antibodies of this invention indetectably labeled form. Antibodies can be detectably labeled throughthe use of radioisotopes, affinity labels (such as biotin, avidin,etc.), enzymatic labels (such as horseradish peroxidase, alkalinephosphatase, etc.) fluorescence labels (such as FITC or rhodamine,etc.), paramagnetic atoms, gold beads, etc. Such labeling procedures arewell-known in the art. The labeled antibodies of the present inventioncan be used for in vitro, in vivo, and in situ assays to identify apolypeptide and/or fragment of this invention in a sample.

In some embodiments, the present invention further provides theabove-described antibodies immobilized on a solid support (e.g., beads,plates, slides or wells formed from materials such as latex orpolystyrene). Examples of such solid supports include plastics such aspolycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir et al., Handbook of Experimental Immunology 4th Ed., BlackwellScientific Publications, Oxford, England, Chapter 10 (1986)). Antibodiescan likewise be conjugated to detectable groups such as radiolabels(e.g., ³⁵S, ¹²⁵I, ¹³¹I), enzyme labels (e.g., horseradish peroxidase,alkaline phosphatase), and fluorescence labels (e.g., fluorescein) inaccordance with known techniques. Determination of the formation of anantibody/antigen complex in the methods of this invention can be bydetection of, for example, precipitation, agglutination, flocculation,radioactivity, color development or change, fluorescence, luminescence,etc., as is well know in the art.

In addition, techniques developed for the production of chimericantibodies or humanized antibodies by splicing mouse antibody genes tohuman antibody genes to obtain a molecule with appropriate antigenspecificity and biological activity can be used (Morrison et al. 1984.Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger et al. 1984. Nature312:604-608; Takeda et al. 1985. Nature 314:452-454). Alternatively,techniques described for the production of single chain antibodies canbe adapted, using methods known in the art, to produce single chainantibodies specific for the polypeptides and fragments of thisinvention. Antibodies with related specificity, but of distinctidiotypic composition, can be generated by chain shuffling from randomcombinatorial immunoglobin libraries (Burton 1991. Proc. Natl. Acad.Sci. 88:11120-3).

Various immunoassays can be used for screening to identify antibodieshaving the desired specificity for the proteins and peptides of thisinvention. Numerous protocols for competitive binding orimmunoradiometric assays using either polyclonal or monoclonalantibodies with established specificity are well known in the art. Suchimmunoassays typically involve the measurement of complex formationbetween an antigen and its specific antibody (e.g., antigen/antibodycomplex formation). For example, a two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on the proteins or peptides of this inventioncan be used, as well as a competitive binding assay.

The present invention is more particularly described in the followingexamples, which are intended as illustrative only since numerousmodifications and variations therein will be apparent to those skilledin the art.

EXAMPLES I. Immunoregulatory Activity of Poxvirus A35R Protein

Cell Tropism.

Several cell types were split into two groups of equal number forinfection. One group was infected with wild-type (Western Reserve or WR)vaccinia virus and the other with the A35R deletion mutant (vacciniavirus that does not contain the A35R protein). Cells were infected at amultiplicity of infection (MOI) of 1. Cells were then incubatedapproximately 20 hours. Three freeze-thaw cycles were then performed torelease newly formed virus particles. Measure of replication wasaccomplished by titering the cell lysates on a monolayer of BS-C-1 cellsand counting the plaques that formed 40 hours later.

Harvest/Infection for Assays.

A 5-9 month old rat (strain: Lewis X from DCM) was injected i.p. with 20μg of C. parvum in 5 ml of HBSS. Three days later the rat was sacrificedand macrophages (peritoneal exudates cells) were harvested. Themacrophages were counted and aliquoted in three 15 ml conical tubesprior to a five hour infection incubation at an MOI of 2. Each treatmentgroup was then washed and plated in a 96 well format. Six 1:2 dilutionswere performed before incubation with 500 nM GPMBP for 30 minutes.25,000 Lewis rat CD4+ RSL-11 clones specific for myelin basic proteinwere added to each well in 80 μl volumes. The plates were incubated for24 hours at 37° C., 5.0% CO₂. After incubation, 150 μl of supernatantwere transferred to an empty 96-well plate and frozen for lateranalysis.

Nitric Oxide Assay.

Fifty microliters of the harvested supernatant were transferred intoanother 96-well plate followed by the addition of Griess Reagent (1%sulfanilamide-0.1% N-[1-naphthy]ethylenediamine in 2.5% phosphoric acid)into each well. The absorbance was read at 540 nm at time intervalsbeginning after a five minute incubation at room temperature. Maximumreadings were achieved after a 1.5-2 hour incubation.

IL-2 Bioassay.

Fifty microliters of the harvested supernatants were transferred intoanother 96-well plate. A total of 50,000 CTLL clones (ATCC TIB-214) werewashed, resuspended in cRPMI, and added to the wells. After 48 hours ofincubation at 37° C., 5.0% CO₂, 10 μl of MTS/PMS was added to everysample. The absorbance was read at 492 nm filtered using 690 nm as thereference wavelength.

VA35Δ Mutant Virus.

To construct the VA35Δ mutant virus, wild-type (WR)-infected cells weretransfected with a recombinant PCR product containing A34R and A36Rflanking sequences with the intervening A35R gene replaced by the E.coli gpt gene. The recombinant vA35Δ was selected in medium containingmycophenolic acid (Roper. 2006. “Characterization of the vaccinia virusA35R protein and its role in virulence” J. Virol. 80:306-313; the entirecontents of which is incorporated by reference herein for studies tocharacterize the poxvirus A35R protein).

Cell Tropism.

The growth of wild-type and A35R deletion mutant virus was studied in 18cell types from six different animals (rats, rabbits, mice, monkeys,hamsters and humans. In one study, the following cell lines were tested:BS-C-1 cells (African green monkey cells), old rat splenocytes, youngrat splenocytes, rat thymocytes, differentiated PC-12 cells,undifferentiated PC-12 cells, activated RSL-11 cells, resting RSL-11cells, PEC, CTLL, brain cells (Heldtilt), HFF cells and R1-T cells. AnA35Δ/wild-type ratio of replication was established. These studiesdemonstrated that PECs, CTLLs and R1-Ts may have tropism where thedeletion mutant had a least a 2-fold greater success replicating thanwild-type. Equal replication was consistent in BS-C-1 cells, which wereused as the control.

Attenuation in Mice.

Four treatment groups (six mice each) of 14-17 g, 5 week-old femaleBALB/c mice were anesthetized and intranasally challenged with 10⁴particle forming units (pfu) of wild type (WR), 35Res (a rescue virusproduced by engineering a wild-type A35R gene back into the vA35Δgenome) or 35Del (the vA35Δ mutant virus), or with PBS. The weight ofthe mice in each group was measured over a period of days. Over a spanof 12 days, the A35R deletion treatment group almost paralleled the PBScontrol group in that gradual weight gain of 10-11% occurred. TheA35-Res and WR groups showed an average weight loss of 16-26%,respectively, by day 12.

Nitric Oxide and IL-2 Bioassay.

Equal volumes of Griess Reagent were added to supernatants to assaynitric oxide in solution. An ELISA plate reader was used to readabsorbance. Infected macrophages secreted less nitric oxide than theuninfected macrophages. However, higher levels of nitric oxide producedby the A35Δ mutant treatment suggests that the presence of the A35Rprotein inhibits secretion of nitric oxide in infected macrophages.Thus, the presence of A35R in macrophages is associated with inhibitingnitric oxide secretion.

Supernatants were assayed using a cytotoxic T lymphocyte line (CTLL-2),which proliferates based on the concentration of IL-2 in solution.MTS/PMS was added to measure respiration dependent on the amount ofproliferation. The absorbance was obtained using an ELISA plate reader.These studies demonstrated that macrophages infected with the A35Δmutant stimulated T cells to secrete more IL-2 in solution than bothuninfected and wild-type (WR) infected cells. This indicates that theA35R protein restricts the ability of macrophages to stimulate T cells.

Additional studies have not demonstrated any effect of A35R on 1)cytotoxic lymphocyte (CTL) killing; 2) interferon-beta (IFN-β) inducedClass I expression in thymocytes; and 3) interferon-gamma (IFN-γ)induced costimulatory molecules (B7.1 molecules) on macrophages.

II. Virulence Activity of Poxvirus A35R Protein

Determination of Virulence in Mice.

Groups of six female BALB/c 6-week-old mice were anesthetized andintranasally challenged with 10⁴ to 10⁶ pfu of vA35Δ, WR, vA35-Resvirus, or PBS in 20 μl of 10 mM Tris-HCl (pH 9.0). Virus titers weredetermined on the day of challenge to confirm virus dose. Individualmice were weighed every two days to monitor health, and mice weresacrificed if they lost 30% of their body weight.

In a second experiment, mice were challenged with 10⁵ and 10⁶ pfu andweighed as described.

The vA35Δ Mutant Virus is Attenuated in Mice.

Over the course of the experiment (14 days), mice challenged with PBS orvA35Δ gained between 10-11% in body weight, whereas mice challenged with10⁴ pfu of the vA35-Res or VV strain WR lost, on average, 16% and 26% oftheir body weight, respectively, by day 10. Two WR-challenged mice (andno others) died between days 9 and 11. This experiment was repeated andattenuation of the vA35Δ was also seen at higher challenge doses. At 10⁵pfu, one mouse challenged with vA35Δ died, compared to four with WR andsix with vA35-Res. When challenged with 10⁶ pfu, all mice died, butsurvival was increased by one to two days in the vA35Δ mice. With WR,two mice died on day 6 and four mice died on day 7. When challenged withvA35-Res, all six mice died on day 6; but with vA35Δ, four mice died onday 7 and two survived until day 8. These data indicate that the A35Rprotein is important for virulence in mice.

Although the present process has been described with reference tospecific details of certain embodiments thereof, it is not intended thatsuch details should be regarded as limitations upon the scope of theinvention except as and to the extent that they are included in theaccompanying claims.

Throughout this application, various patents and non-patent publicationsare referenced. The disclosures of these patents and publications intheir entireties are hereby incorporated by reference into thisapplication in order to more fully describe the state of the art towhich this invention pertains.

TABLE 1 Viral Orthologous Clusters V2.0 (VOCS) Virus data Virus Virusname GenBank Accession No. Vaccinia virus (Tian Tan) VACV-Tan AF095689.1Lumpy skin disease virus (Neethling LSDV-Warm AF409137 Warmbaths LW)Lumpy skin disease virus (Neethling LSDV-1959 AF409138 vaccine LW 1959)Fowlpox virus (HP1-438 Munich) FWPV-Munich AJ581527 Orf virus (NZ2-Sequence 1 ORFV-NZ2_Patent AX754989 from Patent WO03006654) Camelpoxvirus CMS (CMS) CMLV-CMS AY009089 Sheeppox virus (A) SPPV-A AY077833Sheeppox virus (NISKHI) SPPV-NISKHI AY077834 Goatpox virus (G20-LKV)GTPV-G20LKV AY077836 Orf virus (OV-IA82) ORFV-IA82 AY386263 Rabbitpoxvirus (Utrecht) RPXV-Utr AY484669 Vaccinia virus (Acambis 3000 ModifiedVACV-Acambis AY603355 Virus Ankara (MVA)) Monkeypox Virus Walter Reed267 MPXV-WRAIR AY603973 Vaccinia virus (LC16m8) VACV-LC16m8 AY678275Vaccinia virus (Lister) VACV-Lister AY678276 Vaccinia virus (LC16mO)VACV-LC16mO AY678277 Deerpox virus (W-1170-84) DPV-W84 AY689437Monkeypox virus (Sierra Leone) MPXV-SL AY741551 Monkeypox virus (COP-58)MPXV-COP AY753185 Monkeypox virus (USA_2003_044) MPXV-USA_2003_044DQ011153 Monkeypox virus (Congo_2003_358) MPXV-Congo_2003_358 DQ011154Monkeypox virus (Zaire_1979-005) MPXV-Zaire_1979_005 DQ011155 Monkeypoxvirus (Liberia_1970_184) MPXV-Liberia_1970_184 DQ011156 Monkeypox virus(USA_2003_039) MPXV-USA_2003_039 DQ011157 Orf virus (NZ2) ORFV-NZ2DQ184476 Variola major virus (Bangladesh-1975) VARV-Bsh L22579.1 Myxomavirus (Lausanne) MYXV-Laus NC_001132.2 Shope Rabbit fibroma virus(Kasza) SFV-Kas NC_001266 Vaccinia virus (Copenhagen) VACV-CopNC_001559.1 Variola virus (India 3 Major, 1967) VARV-Ind NC_001611.1Molluscum contagiosum virus (subtype 1) MOCV-1 NC_001731.1 Melanoplussanguinipes entomopoxvirus MSEV-Tuc NC_001993.1 (Tucson) Fowlpox virus(Virulent-Iowa) FWPV-Vir_Iowa NC_002188.1 Amsacta moorei entomopoxvirus(Moyer) AMEV NC_002520.1 Yaba-like Disease Virus (Smith) YLDV NC_002642Lumpy skin disease virus (Neethling 2490) LSDV-Neeth NC_003027.1Monkeypox virus (Zaire) MPXV-Zre NC_003310.1 Swinepox virus (Nebraska17077-99) SWPV-Neb NC_003389.1 Camelpox virus (Kazakhstan M-96) CMLV-M96NC_003391.1 Cowpox virus (Brighton Red) CPXV-BR NC_003663.2 Sheeppoxvirus (Turkey-TU-V02127) SPPV-TU NC_004002 Goatpox virus (Pellor)GTPV-Pellor NC_004003 Ectromelia virus (Moscow) ECTV-Mos NC_004105.1Yaba monkey tumor virus YMTV NC_005179 Canarypox virus (ATCC VR-111)CNPV NC_005309 Orf virus (OV-SA00) ORFV-SA00 NC_005336 Bovine papularstomatitis virus (BV-AR02) BPSV-AR02 NC_005337 Deerpox virus (W-848-83)DPV-W83 NC_006966 Vaccinia virus (Western Reserve) VACV-WR NC_006998Ectromelia virus (Naval) ECTV-Nav PBR Vaccinia virus (Modified VacciniaAnkara) VACV-MVA U94848.1 Cowpox virus (GRI-90) CPXV-GRI X94355 Variolaminor virus (Garcia-1966) VARV-Gar Y16780.1 Genes in family “Unknown(Cop-A35R)” GenBank ORF ORF No. of Gene name Accession Virus name startstop amino acids MOCV-1-145R 9629077 MOCV-1 163178 163879 233MYXV-Lau-m123R 9633759 MYXV-Laus 116937 117476 179 SFV-Kas-s123R 6578652SFV-Kas 116088 116627 179 VARV-Gar-A39R 5830703 VARV-Gar 136286 13646860 VARV-Ind-A38R 9627663 VARV-Ind 135262 135444 60 VACV-MVA-146R 2772791VACV-MVA 135418 135948 176 VACV-Cop-A35R 335511 VACV-Cop 143448 143978176 VACV-Tan-TA45R 6969831 VACV-Tan 144144 144674 176 YLDV-124R 12085107YLDV 115059 115598 179 LSDV-Nee-124 15150563 LSDV-Neeth 114604 115179191 MPXV-Zre-A37R 17975060 MPXV-Zre 144036 144566 176 SWPV-Neb-12118640207 SWPV-Neb 112809 113366 185 CMLV-M96-154 18640388 CMLV-M96146790 147320 176 ECTV-Mos-137 22164743 ECTV-Mos 152897 153427 176RPXV-Utr-143 44971506 RPXV-Utr 149272 149805 177 SPPV-TU-119 21492576SPPV-TU 114222 114797 191 CPXV-BR-171 20178533 CPXV-BR 161528 162058 176CMLV-CMS-152R 19718123 CMLV-CMS 144907 145437 176 LSDV-1959-124 22595817LSDV-1959 114440 115015 191 LSDV-Warm-124 22595659 LSDV-Warm 114610115185 191 ECTV-Nav-157 0 ECTV-Nav 150677 151207 176 VACV-WR-15866275955 VACV-WR 144462 144992 176 CPXV-GRI-A36R 30519528 CPXV-GRI160934 161464 176 YMTV-124R 38229287 YMTV 109364 109942 192 ORFV-32167505 ORFV- 111949 112488 179 NZ2_Patent-113 NZ2_Patent BPSV-AR02-11041018863 BPSV-AR02 112527 113081 184 ORFV-AI82-108 41018598 ORFV-IA82111489 112085 198 ORFV-SA00-108 41018731 ORFV-SA00 112723 113262 179VACV-Acambis-148 47088474 VACV-Acambis 129628 130158 176 MPXV-WRAIR-1410 MPXV-WRAIR 144107 144637 176 SPPV-A-119 0 SPPV-A 114198 114773 191SPPV-NISKHI-119 0 SPPV-NISKHI 114001 114576 191 GTPV-Pellor-119 0GTPV-Pellor 113955 114530 191 GTPV-G20LKV-119 0 GTPV-G20LKV 114018114593 191 DPV-W83-134 0 DPV-W83 123533 124078 181 DPV-W84-134 0 DPV-W84126583 127128 181 VACV-LC16m8- 56713552 VACV-LC16m8 147115 147645 176m8202R VACV-LC16mO- 56713836 VACV-LC16mO 147114 147644 176 mO202RVACV-Lister- 0 VACV-Lister 146991 147521 176 m8202R MPXV-COP-14159858947 MPXV-COP 144350 144880 176 MPXV-SL-141 58220611 MPXV-SL 144152144682 176 MPXV- 68448826 MPXV- 144173 144703 176 USA_2003_044-USA_2003_044 155 MPXV- 68449229 MPXV- 144675 176 Zaire_1979_005-Zaire_(——)1979_010454145 155 MPXV- 68449027 MPXV- 144706 176Congo_2003_358- Congo_2003_315484176 155 MPXV- 68449428 MPXV- 145126 176Liberia_1970_184- Liberia_1970_114844596 155 MPXV- 68449628 MPXV- 144173144703 176 USA_2003_039- USA_2003_039 155 ORFV-NZ2-111 74230824 ORFV-NZ2111955 112494 179

TABLE 2

In Table 2: BPSV, Bovine popular stomatitis virus (SEQ ID NO: 8) CMLV,Camelpox virus (SEQ ID NO: 9) CPXV, Cowpox virus (SEQ ID NO: 10) DPV,Deerpox virus (SEQ ID NO: 11) ECTV, Ectromelia virus (SEQ ID NO: 12)GTPV, Goatpox virus Pellor (SEQ ID NO: 13) LSDV, Lumpy skin diseasevirus (SEQ ID NO: 14) MOCV, Molluscum contagiosum virus (SEQ ID NO: 15)MPXV, Monkeypox virus (SEQ ID NOs: 16, 17 and 18) MYXV, Myxoma virus(SEQ ID NO: 19) ORFV, Orf virus (SEQ ID NO: 20) RPXV, Rabbitpox virus(SEQ ID NO: 21) SFV, Rabbit fibroma virus (SEQ ID NO: 22) SPPV, Sheeppoxvirus (SEQ ID NO: 23) SWPV, Swinepox virus (SEQ ID NO: 24) VACV,Vaccinia virus (SEQ ID NOS: 25, 26, 27, 28 and 29) YLDV, Yaba-likedisease virus (SEQ ID NO: 30) YMTV, Yaba-like monkey tumor virus (SEQ IDNO: 31)

TABLE 3

In Table 3: BPSV, Bovine popular stomatitis virus (SEQ ID NO: 32) DPV,Deerpox virus (SEQ ID NO: 33) LSDV, Lumpy skin disease virus (SEQ ID NO:34) MOCV, Molluscum contagiosum virus (SEQ ID NO: 35) MYXV, Myxoma virus(SEQ ID NO: 36) ORFV, Orf virus (SEQ ID NO: 37) SPPV Sheeppox virus (SEQID NO: 38) SWPV, Swinepox virus (SEQ ID NO: 39) VACV, Vaccinia virus(SEQ ID NO: 40) VARY, Variola virus (SEQ ID NO: 41) YLDV, Yaba-likedisease virus (SEQ ID NO: 42) YMTV, Yaba-like monkey tumor virus (SEQ IDNO: 43)

1. A method of modulating an immune response in a subject, comprisingadministering to the subject an effective amount of an A35R protein oractive fragment thereof of vaccinia virus or other poxvirus. 2-29.(canceled)